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

Figure 4.

Figure 4.

A CP110 mutant unable to bind to HOIP fails to be removed from the mother centrioles and inhibits ciliogenesis. (A) Schematic of the interaction region of CP110 with HOIP. +, interaction, −, no interaction. (B) The interaction of Flag-CP110 WT and Δ832–991 mutant with Myc-HOIP. HEK293T cells were transfected with Myc-HOIP together with Flag-vector (−), Flag-CP110 WT, or Δ832–991 mutant. The cell lysates were immunoprecipitated with ANTI-FLAG M2 Affinity Gel, then the immunoprecipitates and cell lysates were detected by immunoblotting with indicated antibodies. (C) Linear ubiquitination assay of Flag-CP110 WT and Δ832–991 mutant in HEK293T cells cotransfected with LUBAC components. (D) RPE-1 stable cell line expressing Flag-CP110 WT and Δ832–991 mutant were transfected with control or CP110 siRNAs and serum starved for 48 h. The cells were stained with the indicated antibodies. Scale bar, 1 µm. (E) Quantification of cells with one CP110 dot in Flag-positive RPE-1 cells in D. (F) Quantification of the ciliated cells in Flag-positive RPE-1 cells in D. Data are presented as mean ± SD of three independent experiments in E and F. ***, P < 0.001 by unpaired two-tailed t test. FL, full-length; IB, immunoblot; IP, immunoprecipitation; tub, tubulin; Ub, ubiquitin; (Linear Ub)n, poly-linear ubiquitin chains.