PMC6393651_01
Intestinal current measurements of four rectal suction biopsies collected from (a) the elder (upper panel) and (b) the younger (lower panel) p.Phe508del/p.[Arg74;Val201Met;Asp1270Asn] compound heterozygous siblings. The original tracings show the responses (from left to right) to amiloride, forskolin/IBMX, genistein, carbachol, 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and histamine. Please note that the measurements were performed in Mini-Ussing chambers with tissue sliders with an aperture area of either 0.018 or 0.011 cm2. The registration of the blue tracing in (a) became invalid after exposure to DIDS because of technical reasons (gray bar)
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In contrast, the transient pulses of chloride secretion evoked in the biopsies by basolateral Ca2+-dependent K+ efflux induced by carbachol or histamine (Bronsveld et al., 2000) were donor-dependent either in the normal or in the CF range (Figure 1).
CFTR immunoblot. (a) Immunodetection of CFTR (from left to right) in lysates of rectal suction biopsies by immunoprecipitation (IP) and subsequent immunoblot from a healthy non-CF subject (lane 2) and its IP bead control (lane 1), the elder p.Phe508del/p.[Arg74;Val201Met;Asp1270Asn] compound heterozygous index case (lane 3) and its IP bead control (lane 4) and in lysates of T84 cells (lane 5). The right panel shows electronically enhanced signals of lanes 2 and 3 of the blot in order to more clearly visualize the CFTR immunoreactive signals of the biopsies. (b) Immunodetection of CFTR by immunoblot of lysates of rectal suction biopsies of two healthy non-CF subjects (lanes 1, 3; 87 microg each) and of the younger p.Phe508del/p.[Arg74;Val201Met;Asp1270Asn] compound heterozygous index case (lane 2; 87 microg) (lane 1-3) and of non-CF 16HBE14o cells (lane 4, 15 microg; lane 5, 35 microg; lane 6, 50 microg). Vinculin signals are provided as a western blot loading control
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Next, we determined the CFTR protein levels in the biopsies (Figure 2). The immunoblot detected fourfold lower amounts of complex glycosylated CFTR in immunoprecipitates of rectal biopsies collected from the elder sibling than in specimens taken from wild-type controls (Figure 2a). In contrast, the intensity of immunoreactive CFTR C- and B-bands was indistinguishable between samples from the younger sibling and non-CF controls (Figure 2b).