HeLa lysate assay. Immobilized protein activity was also assessed using phosphorylation of endogenous substrates in a HeLa cell lysate. Briefly, 50 µL of resuspended beads were combined with 50 µL of a HeLa cell lysate (0.140 mg/mL, prepared from cultured cells lysed with M-PER + 1X HALT protease and phosphatase inhibitors and stored at -80 °C). The samples were incubated at for 1h at room temperature on an end-over-end rotator. Following incubation, beads were pelleted and the supernatants were transferred to clean tubes. 6X Laemmli sample buffer (20 µL) was added to each sample, followed by heating at 65 °C for 10 min. and subsequently analyzed via western blot as previously described (PKA pS/T substrate antibody (Cell Signaling Technologies #9624S); 1:1000 dilution in TBST + 5% BSA).