Figure 4.

MIB1 promotes ubiquitylation of centriolar satellite factors and is inactivated by cell stress. (A) SILAC-labelled U2OS/Strep-HA-ubiquitin cells were mock treated (Light (L) medium) or subjected to UV radiation (25 J/m²) or ionizing radiation (IR, 10 Gy) (Heavy (H) medium) and collected 1 h later. Ubiquitylated proteins were purified on Strep-Tactin resin under denaturing conditions and analysed by mass spectrometry (MS) for determination of SILAC ratios for individual proteins (Supplementary Figure S4A; Supplementary Table S1). SILAC ratios (H/L), indicating relative DNA damage-induced change in ubiquitylation level, for selected proteins are shown. (B) U2OS/Strep-HA-ubiquitin cells left untreated or exposed to UV were collected 1 h later. Where indicated, p38 inhibitor (p38i) was added to the medium 30 min before UV radiation. Cell extracts were subjected to Strep-Tactin pull-down followed by immunoblotting with MIB1 antibody. (C) U2OS or U2OS/Strep-HA-ubiquitin cells were mock treated or subjected to UV, collected 1 h later, and lysed under denaturing conditions. Ubiquitylation of centriolar satellite proteins was analysed by immunoblotting Strep-Tactin pull-downs from whole-cell extracts (WCEs) with AZI1, PCM1, and CEP290 antibodies. (D) U2OS or U2OS/Strep-HA-ubiquitin cells were transfected for 72 h with control siRNA or independent siRNAs targeting MIB1. Ubiquitylation of AZI1 and PCM1 in WCEs was analysed by immunoblotting Strep-Tactin pull-downs with the indicated antibodies. (E) U2OS or U2OS/Strep-HA-ubiquitin cells were transfected for 24 h with WT or catalytically inactive (CI) versions of GFP-MIB1 and treated or not with UV. Ubiquitylation of AZI1 and PCM1 was analysed as in (C). (F) Extracts of U2OS cells transfected with indicated combinations of plasmids were analysed for ubiquitylation of AZI1(F1) (amino acids 1–268) by immunoblotting S-tag pull-downs with HA antibody.