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. 2009 Apr 10;106(16):6650–6655. doi: 10.1073/pnas.0901083106

Fig. 4.

Fig. 4.

USP33 knockdown prevents β-arrestin deubiquitination, promotes endosomal trafficking, and prolongs ERK signaling. (A) HEK-293 cells were transfected with HA-β2AR, β-arrestin2-Flag, and either control or USP33 siRNA. Flag immunoprecipitates were isolated after isoproterenol stimulation for the indicated times and probed with an anti-ubiquitin antibody (Upper). (Lower) USP33 levels in control and USP-knockdown samples. (B) Confocal micrographs show distribution of Flag-β2AR (red) and β-arrestin2-GFP (green) in USP33-depleted cells (Upper) and control (Lower) cells in agonist-stimulated HEK-293 cells. (Scale bars, 10 μm.) (C) Western blots of p-ERK and ERK in response to a time course of isoproterenol (100 nM) activation, detected in HEK-293 cells with stable β2AR expression (2 pmol/mg of protein) and with indicated siRNA transfections. (D) Quantification of p-ERK signals from 3 independent experiments. **, P < 0.01, control versus USP33, 2-way ANOVA. (E) A representative blot for USP33 levels showing Western analysis of 25 μg of lysate protein from each siRNA transfection.