SDS–PAGE was done using homemade Bis-acrylamide gels or Mini-PROTEAN TGX gradient gels (Bio-Rad). Gels were blotted onto PVDF membranes. Membranes were blocked in 5% milk powder or 10% Roti-Block (Roth). Primary antibodies were added overnight at 4°C in 5% milk powder or 10% Roti-Block (Roth)—1:1,000 Ub antibody P4D4 (Santa Cruz Biotechnology), 1:3,000 Hip1 (22231-1AP; Proteintech), 1:000 PARP10/ARD10 (NB100-2157; Novus Biologicals), 1:1,000 Rad23b (E-AB-62188; Elabscience), 1:1,000 Dsk2 (ab4119-100; Abcam), 1:4,000 Rad23 (Sommer Lab), 1:1,000 Epn2 (Invitrogen), DDI2 (AB197081; Abcam), 1:1,000 DDI1 serum (Jeffrey Gerst, Weizmann Institute of Science (99 (link))), 1:1,000 APPL1 (D83H4; Cell Signaling), 1:1,000 CCDC50 (AB127169; Abcam), 1:1,000 FAF1 (1027-1-AP; Proteintech), 1:1,000 ZFAND2B (Sigma-Aldrich), 1:1,000 Riok3 (13593-1-AP; Proteintech), 1:1,000 USP11 (22340-1-AP; Proteintech), 1:5,000 CDC48 (Sommer Lab), 1:1,000 Vps9 (Scott Emr, Cornell University (100 (link))), 1:1000 YUH1 serum (Tingting Yao, Colorado State (101 (link))). Anti-mouse IgG HRP (Sigma-Aldrich) and anti-rabbit IgG HRP (Sigma-Aldrich) were used at 1:10,000 as secondary antibodies. Immunoblots were visualized using an Odyssey XF imager (LI-COR).
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