Fig. 7
From: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
EGFR directly phosphorylates USP8 on Tyr-717 and Tyr-810 to elevate its DUB activity. a Anti-EGFR immunoprecipitates from serum-fed (serum: + ) or -starved (serum: -) RPE1 cell lysates were analyzed by anti-USP8 and anti-EGFR immunoblotting. b Bacterially purified GST-USP8 was incubated with purified GST-EGFR (669–1210 aa) in the presence or absence of 1 μM PD153035 for 15 min at 30 °C. c Bacterially purified non-tagged USP8 variants (WT, Y717F/Y810F, Y717F, and Y810F) were incubated with or without GST-EGFR (669–1210 aa) for 15 min at 30 °C, and then incubated with ubiquitin oligomers (Ub3–7) for 15 min at 37 °C. d TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F) cells treated with 10 ng ml−1 of Dox (serum: + ) were subjected to 24 h serum starvation (serum: −). Anti-pY and anti-FLAG immunoblotting of anti-FLAG immunoprecipitates are shown. e, f TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F; YF) cells were transfected with control or USP8 siRNA (#1), and then cultured for 48 h in the presence of indicated concentration of Dox. Representative confocal images of acetylated-tubulin (green), FLAG (red) and DAPI (blue) are shown in e. Scale bar, 20 μm. Normalized intensities of trichoplein/GAPDH are calculated by immunoblotting (f) and shown as mean from three independent biological replicates. Percentages of ciliated cell (mean ± SD from three independent experiments, n > 200 each) are shown in f. Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. **p < 0.01, *0.01 < p < 0.05, n.s., not significant, two-tailed unpaired student’s t-tests
