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<article doi="10.1002/eji.200737900" abstract="Accumulating evidence suggests that intracellular antigens are endogenously presented on MHC class II, but it is still unknown whether antigens within different subcellular compartments are presented with similar efficiency, and via the same or different pathways. We have previously shown that endogenous MHC class II presentation of the cytosolic bacterial antigen neomycin phosphotransferase II (NeoR) is mediated by autophagy. Here, we addressed whether secluding NeoR from this cytoplasmic pathway by directing the protein into the cell nucleus (NucNeoR) would affect antigen presentation. Unexpectedly, NucNeoR was presented at least as efficiently as the cytosolic version of the antigen. Furthermore, presentation of NucNeoR was also dependent on autophagocytosis and lysosomal processing, indicating that both antigens were presented via the same pathway. Inhibition of CRM1-mediated nuclear export did not impede antigen presentation, indicating that NucNeoR gained access to this autophagy-dependent MHC class II presentation pathway by a CRM1-independent route. Thus, this endogenous presentation pathway broadens the spectrum of intracellular antigens surveyed by CD4(+) T cells by efficiently sampling cytoplasmic as well as nuclear antigens."><fig id="716"><title></title><label>Figure 1</label><graphic href="https://api.sourcedata.io/file.php?figure_id=716"/><sd-panel panel_id="998"><p>Subcellular localization of <sd-pretag id="sdPretag14237289255650" parent-tag-id="7" class="intervention protein">NeoR</sd-pretag> and NucNeoR and T cell recognition of <sd-pretag id="sdPretag14237289255651" parent-tag-id="7" class="intervention protein">NeoR</sd-pretag>‐ and NucNeoR‐expressing cells. <sd-tag id="sdTag6" source="sdapi" category="entity" entity_type="cell" role="component" text="RCC1.24 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">RCC1.24 cells</sd-tag> were transfected with the <sd-tag id="sdTag7" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by <sd-tag id="sdTag16" source="sdapi" category="assay" entity_type="" role="" text="immunofluorescence" ext_ids="BAO_0000450" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="fluorescence microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">immunofluorescence</sd-tag> using the anti <sd-tag id="sdTag9" source="sdapi" category="entity" entity_type="protein" role="reporter" text="c‐myc" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">c‐myc</sd-tag> antibody 9E10. While <sd-tag id="sdTag10" source="sdapi" category="entity" entity_type="protein" role="assayed" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> was evenly distributed in <sd-tag id="sdTag11" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="nucleus" ext_ids="GO:0005634" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="nucleus" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">nucleus</sd-tag> and <sd-tag id="sdTag12" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="cytoplasm" ext_ids="GO:0005737" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="cytoplasm" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">cytoplasm</sd-tag> (a), NucNeoR was confined to the cell <sd-tag id="sdTag14" source="sdapi" category="entity" entity_type="subcellular" role="experiment" text="nucleus" ext_ids="GO:0005634" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="nucleus" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">nucleus</sd-tag> (b). <sd-tag id="sdTag15" source="sdapi" category="assay" entity_type="" role="" text="Daylight photographs" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Daylight photographs</sd-tag> of the stained cells are shown in (a') and (b').</p><graphic href="https://api.sourcedata.io/file.php?panel_id=998"/></sd-panel><sd-panel panel_id="999"><b>(C)</b> <sd-tag id="sdTag17" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag>‐ and NucNeoR‐transfected <sd-tag id="sdTag19" source="sdapi" category="entity" entity_type="cell" role="component" text="RCC1.24 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls=""><sd-pretag id="sdPretag14236671549170" parent-tag-id="57" class="component cell">RCC1.24</sd-pretag> cells</sd-tag> were treated with 100 U/mL <sd-tag id="sdTag20" source="sdapi" category="entity" entity_type="protein" role="intervention" text="IFN‐γ" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">IFN‐γ</sd-tag> for 48 h to induce <sd-tag id="sdTag21" source="sdapi" category="entity" entity_type="protein" role="component" text="MHC class II" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">MHC class II</sd-tag> expression. Subsequently, the cells were washed and co‐cultured with the <sd-tag id="sdTag156" source="sdapi" category="entity" entity_type="cell" role="component" text="CD4 clone 20‐4/A4" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">CD4 clone 20‐4/A4</sd-tag>, which recognizes a peptide derived from <sd-tag id="sdTag24" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> on <sd-tag id="sdTag25" source="sdapi" category="entity" entity_type="protein" role="component" text="HLA‐DP3" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">HLA‐DP3</sd-tag>. <sd-tag id="sdTag26" source="sdapi" category="entity" entity_type="protein" role="assayed" text="GM‐CSF" ext_ids="P04141" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="CSF2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">GM‐CSF</sd-tag> secretion by the <sd-pretag id="sdPretag14236688669000" parent-tag-id="149">T cells</sd-pretag> was measured 20 h later by <sd-tag id="sdTag27" source="sdapi" category="assay" entity_type="" role="" text="ELISA" ext_ids="BAO_0000134" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="ELISA" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">ELISA</sd-tag>. Both transfectants were recognized after <sd-tag id="sdTag28" source="sdapi" category="entity" entity_type="protein" role="intervention" text="IFN‐γ" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">IFN‐γ</sd-tag> treatment, indicating that directing <sd-tag id="sdTag29" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> into the cell nucleus does not abrogate antigen presentation.<graphic href="https://api.sourcedata.io/file.php?panel_id=999"/></sd-panel><sd-panel panel_id="1000"><b>(D)</b> <sd-tag id="sdTag31" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> were transfected with <sd-tag id="sdTag32" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the <sd-tag id="sdTag34" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag>‐specific <sd-tag id="sdTag157" source="sdapi" category="entity" entity_type="cell" role="component" text="T cell clone 20‐4/A4" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">T cell clone 20‐4/A4</sd-tag>. Antigen‐transfected <sd-tag id="sdTag37" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> were recognized irrespectively of <sd-tag id="sdTag38" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> localization, while untransfected cells or cells transfected with a <sd-tag id="sdTag39" source="sdapi" category="entity" entity_type="protein" role="normalizing" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐expressing control plasmid were not recognized.<graphic href="https://api.sourcedata.io/file.php?panel_id=1000"/></sd-panel></fig><fig id="717"><title></title><label>Figure 2</label><graphic href="https://api.sourcedata.io/file.php?figure_id=717"/><sd-panel panel_id="1001"><b>(A)</b> <sd-tag id="sdTag57" source="sdapi" category="entity" entity_type="cell" role="component" text="RCC1.24" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">RCC1.24</sd-tag>‐<sd-tag id="sdTag50" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and ‐NucNeoR cells were incubated for 48 h with <sd-tag id="sdTag52" source="sdapi" category="entity" entity_type="protein" role="intervention" text="IFN‐γ" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">IFN‐γ</sd-tag> and in addition with 7.5 mM <sd-tag id="sdTag58" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="3‐MA" ext_ids="CHEBI:38635" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="3-methyladenine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">3‐MA</sd-tag> or 200 µM <sd-tag id="sdTag59" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="leupeptin" ext_ids="CHEBI:6426" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="Leupeptin" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">leupeptin</sd-tag> or 100 µM <sd-tag id="sdTag60" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="chloroquine" ext_ids="CHEBI:3638" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="chloroquine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">chloroquine</sd-tag> for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with <sd-tag id="sdTag158" source="sdapi" category="entity" entity_type="cell" role="component" text="20‐4/A4 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">20‐4/A4 cells</sd-tag> in a <sd-tag id="sdTag54" source="sdapi" category="entity" entity_type="protein" role="assayed" text="GM‐CSF" ext_ids="P04141" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="CSF2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">GM‐CSF</sd-tag> release assay. Both <sd-tag id="sdTag55" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.<graphic href="https://api.sourcedata.io/file.php?panel_id=1001"/></sd-panel><sd-panel panel_id="1002"><b>(B)</b> <sd-tag id="sdTag62" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> transfected with <sd-tag id="sdTag63" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> or NucNeoR expression plasmids were tested likewise. Presentation of endogenous <sd-tag id="sdTag65" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and NucNeoR was blocked by all inhibitors, indicating that <sd-tag id="sdTag67" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and NucNeoR presentation is dependent on autophagy and lysosomal processing.<graphic href="https://api.sourcedata.io/file.php?panel_id=1002"/></sd-panel><sd-panel panel_id="1003"><b>(C)</b> To exclude toxic side effects of the inhibitors, <sd-tag id="sdTag89" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> were incubated for 20 h with 200 ng/mL recombinant <sd-tag id="sdTag90" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the <sd-tag id="sdTag91" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag>‐specific <sd-tag id="sdTag99" source="sdapi" category="entity" entity_type="cell" role="component" text="CD4+ T cell clone 20‐4/A4" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">CD4<sup>+</sup> T cell clone 20‐4/A4</sd-tag>. The presentation of exogenous <sd-tag id="sdTag94" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> on <sd-tag id="sdTag98" source="sdapi" category="entity" entity_type="protein" role="component" text="MHC class II" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">MHC class II</sd-tag> was not affected by <sd-tag id="sdTag109" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="3‐MA" ext_ids="CHEBI:38635" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="3-methyladenine" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">3‐MA</sd-tag>, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.<graphic href="https://api.sourcedata.io/file.php?panel_id=1003"/></sd-panel><sd-panel panel_id="1004"><b>(D)</b> <sd-tag id="sdTag110" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> were transfected with siRNA specific for <sd-tag id="sdTag112" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Atg12" ext_ids="9140" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg12</sd-tag> or <sd-tag id="sdTag111" source="sdapi" category="entity" entity_type="gene" role="normalizing" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>, and <sd-tag id="sdTag113" source="sdapi" category="entity" entity_type="gene" role="assayed" text="Atg12" ext_ids="9140" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg12</sd-tag> mRNA levels were determined 24 h later by <sd-tag id="sdTag115" source="sdapi" category="assay" entity_type="" role="" text="qPCR" ext_ids="BAO_0002084" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="real-time PCR " ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">qPCR</sd-tag>. The relative amount of <sd-tag id="sdTag114" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Atg12" ext_ids="9140" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg12</sd-tag> mRNA was reduced approximately fourfold.<graphic href="https://api.sourcedata.io/file.php?panel_id=1004"/></sd-panel><sd-panel panel_id="1005"><b>(E)</b> At 24 h after siRNA transfection, <sd-tag id="sdTag118" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with <sd-tag id="sdTag120" source="sdapi" category="entity" entity_type="gene" role="intervention" text="Atg12" ext_ids="9140" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="ATG12" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">Atg12</sd-tag> siRNA, but not in mock‐transfected cells or cells transfected with <sd-tag id="sdTag121" source="sdapi" category="entity" entity_type="gene" role="normalizing" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag> siRNA, antigen presentation of both <sd-tag id="sdTag122" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> and NucNeoR was greatly reduced.<graphic href="https://api.sourcedata.io/file.php?panel_id=1005"/></sd-panel></fig><fig id="718"><title></title><label>Figure 3</label><graphic href="https://api.sourcedata.io/file.php?figure_id=718"/><sd-panel panel_id="1006"><b>(A)</b> <sd-tag id="sdTag131" source="sdapi" category="entity" entity_type="cell" role="component" text="RCC1.24 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">RCC1.24 cells</sd-tag> were transfected with the NES‐<sd-tag id="sdTag133" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐<sd-tag id="sdTag134" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag>‐<sd-tag id="sdTag135" source="sdapi" category="entity" entity_type="protein" role="reporter" text="GFP" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">GFP</sd-tag>‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM <sd-tag id="sdTag138" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="LMB" ext_ids="CHEBI:52646" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="leptomycin B" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">LMB</sd-tag> for 20 h (b) showed a strong accumulation of the protein in the <sd-tag id="sdTag136" source="sdapi" category="entity" entity_type="subcellular" role="component" text="nucleus" ext_ids="GO:0005634" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="nucleus" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">nucleus</sd-tag>. (a') and (b') are the corresponding <sd-tag id="sdTag139" source="sdapi" category="assay" entity_type="" role="" text="daylight photograph" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">daylight photograph</sd-tag>s of the transfected cells.<graphic href="https://api.sourcedata.io/file.php?panel_id=1006"/></sd-panel><sd-panel panel_id="1007"><b>(B)</b> <sd-tag id="sdTag140" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag>‐ and NucNeoR‐transfected <sd-tag id="sdTag142" source="sdapi" category="entity" entity_type="cell" role="component" text="LCL1.11 cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">LCL1.11 cells</sd-tag> were either left untreated or incubated for 20 h with 8 nM <sd-tag id="sdTag143" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="LMB" ext_ids="CHEBI:52646" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="leptomycin B" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">LMB</sd-tag>. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the <sd-tag id="sdTag151" source="sdapi" category="entity" entity_type="cell" role="component" text="NeoR‐specific T cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR‐specific T cells</sd-tag>. <sd-tag id="sdTag145" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="LMB" ext_ids="CHEBI:52646" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="leptomycin B" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">LMB</sd-tag> treatment had no significant effect on the presentation of <sd-tag id="sdTag146" source="sdapi" category="entity" entity_type="protein" role="intervention" text="NeoR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">NeoR</sd-tag> or NucNeoR on <sd-tag id="sdTag148" source="sdapi" category="entity" entity_type="protein" role="component" text="MHC class II" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">MHC class II</sd-tag>, indicating that <sd-tag id="sdTag152" source="sdapi" category="entity" entity_type="protein" role="component" text="CRM1" ext_ids="O14980" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="XPO1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">CRM1</sd-tag>‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.<graphic href="https://api.sourcedata.io/file.php?panel_id=1007"/></sd-panel></fig></article> |
