Hpa021578

The primary antibodies used were as follows: TRIM65 (1:1000, HPA021578, Sigma-Aldrich), GAPDH (1:10000, 60004-1-Ig, Proteintech), and ARHGAP35 (GRLF1, 1:1000, 26789-1-AP, Proteintech). The secondary antibodies were anti-rabbit IgG-HRP and anti-mouse IgG-HRP (7074 S and 7076, Cell Signaling Technology). Other antibodies are listed in Supplemental file.

Proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes (GE Healthcare) using wet blot transfer. Total protein was stained using Ponceau S and membranes were blocked and incubated with antibodies either in 5% milk or 5% BSA in TBS (150 mM NaCl and 20 mM Tris pH 8.0). Washes were performed in TBS-T (TBS and 0.1% Tween20). After incubation with horesradish peroxidase-conjugated secondary antibodies, membranes were developed using chemiluminescence reagents ImmunoCruz (Santa Cruz) or TMA-6 (Lumigen). The following commercial primary antibodies were used: α-HA (16B12, Covance MMS-101, 1:1,000), α-GFP (B-2, Santa Cruz sc-9996, 1:1,000), α-FLAG (M2, Sigma F3165, 1:5,000), α-Ub (P4D1, Santa Cruz sc-8017, 1:1,000), α-TRIM56 (Abcam ab154862, 1:10,000), α-TRIM65 (Sigma HPA021578, 1:500), α-Tubulin (Sigma T9026, 1:5,000) and α-Vinculin (Sigma hVIN-1, 1:2,000). Uncropped scans of developed membranes are provided in Supplementary Figs 6,7,8 and 9 as part of the Supplementary Information.