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. 2021 Nov 23;221(1):e202105092. doi: 10.1083/jcb.202105092

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© 2021 Shen et al.

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Figure 5. — PRPF8 is a receptor for the linear ubiquitin chains of CP110 and promotes CP110 removal during ciliogenesis. (A) RPE-1 cells were serum starved for 8 h, then the cell lysates were pulled down by GST or GST-Ub4, with GST as a negative control. Endogenous PRPF8 was detected by anti-PRPF8 antibody. GST and GST-Ub4 were stained by Ponceau S. (B) Flag-CP110 was transfected into HEK293T cells with or without LUBAC WT or C885S mutant. The cell lysates were immunoprecipitated with ANTI-FLAG M2 Affinity Gel, and then the immunoprecipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) RPE-1 cells were transfected with control or two individual PRPF8 siRNAs followed by serum starvation for 48 h. The cells were stained with the indicated antibodies. Scale bars, 1 µm. (D) Quantification of cells with one CP110 dot in RPE-1 cells in C. (E) Immunoblot of the RPE-1 cell lysates in C with the indicated antibodies. (F) Knockdown of CP110 rescued the defective ciliogenesis in PRPF8-depleted cells. RPE-1 cells were transfected with PRPF8 or CP110 siRNAs or cotransfected with PRPF8 and CP110 siRNAs. Cells then were serum starved for 48 h and stained with ARL13B (red). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 2 µm (magnified region). (G) Quantification of the ciliated cells in F. (H) Immunoblot of the RPE-1 cell lysates in F with the indicated antibodies. Data are presented as mean ± SD of three independent experiments in D and G. ***, P < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test in D and Bonferroni’s multiple comparisons test in G. Ctrl, control; IB, immunoblot; IP, immunoprecipitation; tub, tubulin; Ub, ubiquitin; (Linear Ub)_n, poly-linear ubiquitin chains.