Sc 25351

Cells were lysed with Laemmli sample buffer (5% SDS, 25% glycerol, 150 mM Tris-HCl (pH 6.8) and 0.01% bromophenol blue). After protein concentration was measured by BCA protein assay, 0.7 M β-mercaptoethanol was added and protein samples were boiled for 10 min. Protein samples were separated by SDS–PAGE, and protein was transferred onto a polyvinylidene fluoride membrane (Immobilon-P membrane, Millipore). Specific antibodies to ALDH1A1 (1:1,000, 611194, BD Bioscience), SAE2 (1:1,000, ab58451, Abcam), SUMO-2,3 (1:500, M114-3, MBL), Ubc9 (1:1,000, #4918, Cell Signaling Technology, Inc.), Oct-1 (1:1,000, #8517, Cell Signaling Technology, Inc.), ubiquitin (1:1,000, MAB1510, Millipore), TRIM21 (1:1,000, sc-25351, Santa Cruz), IRF1 (1:1,000, #8478, Cell Signaling Technology, Inc.), SUMO-1 (1:500, #4930, Cell Signaling Technology, Inc.), SENP2 (1:1,000, sc-67075, Santa Cruz Biotech), PIAS1 (1:1,000, #3550, Cell Signaling Technology, Inc.) and GAPDH (1:1,000, sc-20357, Santa Cruz) were detected using the appropriate secondary antibodies (Licor) and visualized by Odyssey detection system (Licor). The uncropped scans of western blots are provided as Supplementary Fig. 12.

Cell lysates were electrophoresed on Nu-PAGE gels (Life Technologies) and transferred to nitrocellulose membranes using the iBlot rapid transfer system (Life Technologies). Antibodies to human TRIM21 (Sc-25351, Santa Cruz) with HRP-conjugated anti-mouse (A0168, Sigma), and HRP-conjugated beta-Actin (Sc-47778, Santa Cruz) were used to detect protein levels.