Anti uba2

Cells were washed with PBS and then lysed with either the lysis buffer (#9803; Cell Signaling Technology; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na₃VO₄, 1 μg/mL leupeptin) or the acidic lysis buffer (50 mM HEPES, pH 6.0, 150 mM NaCl, 0.1% (w/v) SDS) supplemented with protease cocktail (Roche). The acidic lysis buffer was used to preserve the ubiquitin or ubiquitin-like conjugations (e.g., UBA3^N8, Ubc12^N8, UbcH7^Ub, Ubc9^SUMO) that are labile in the regular lysis buffer. The supernatant of the lysate was used for Western blotting. The antibodies used in this study include anti-cullin1 (H213; Santa cruz), anti-UBA3 (F-10; Santa cruz), anti-GlyRS (B01P; Abnova or sc-98614; Santa cruz), anti-SerRS (homemade), anti-V5 (R96-CUS; Invitrogen), and anti-Ube2F (pa5-26641; Thermo Fisher). Other antibodies including anti-NEDD8 (#2754), anti-Ubiquitin (#3936), anti-SUMO1 (#4930), anti-Ubc12 (#5641), anti-Ubc9 (#4918), anti-Flag (#2908), anti-UBA1(#4891), anti-UbcH7 (#3848), anti-UBA2 (#8688), anti-APPBP1 (#14321), anti-p27kip (#3698), and anti-α-Tubulin (#3873) are from Cell Signaling.