Figure 1.

USP8 depletion induces abnormal accumulation of K63-linked ubiquitinated plasma membrane proteins on endosomes. (A and B) HeLa cells were transfected with the indicated siRNAs. The absolute abundance of K48 and K63 ubiquitin chains in total cell lysates (A) or membrane fractions (B) was analyzed by Ub-PRM. Error bars indicate SD (total cell lysate n = 2 in A, membrane fraction n = 4 in B). *P < 0.05 and **P < 0.01 (two-tailed Student’s t test). (C) HeLa cells transfected with the indicated siRNAs were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (D) Experimental procedures are shown (see also Materials and methods section). HeLa cells transfected with the indicated siRNAs were subjected to LC-MS/MS analysis. (E) The mean log₂ FC (siUSP8/siControl) and −log₁₀ P value of the ubiquitin conjugates analysis are shown on the x- and y-axis, respectively. (F) The bubble plot illustrating the GOCC of ubiquitinated proteins increased in USP8-depleted cells is shown (top 10 categories). (G) The heatmap illustrates the changes of proteins hit in the plasma membrane in F. Colors indicate the scaled intensity.