Rpn2106

Western blot analysis was performed as previously described [28]. Cells and tissues were lysed in Laemmli buffer; for tissues samples were homogenised with the Precellys 24 tissue homogeniser (Precellys) in Laemmeli buffer. Samples were run on 12.5% or 15% gels and transferred to PVDF membranes (Immobilon: Millipore, IPVH00010). The membrane was blocked for 1 h at room temperature with 5% milk solution in TBS-Tween (Tris-buffered saline [50 mM Tris, 150 mM NaCl, pH 8.0] containing 0.1% Tween 20 [Fisher BP337-500]) before incubating with primary antibody at 4°C overnight. Subsequently, an appropriate HRP-conjugated secondary antibody was added followed by incubation at room temperature for 1 h. Western blots were visualised with chemiluminsence reagents (Sigma, RPN2106).

Tissue samples were homogenised with the Precellys 24 tissue homogeniser in Laemmli buffer and samples ran on 12.5 or 15% gels. Protein was transferred to PVDF membranes (Immobilon, Millipore), which was subsequently blocked for 1 h at room temperature (5% milk solution in TBS-Tween 0.1%) before incubating with primary antibody at 4 °C overnight. An appropriate HRP-conjugated secondary antibody was incubated at room temperature for 1 h. Western blots were visualised with chemiluminescence reagents (Sigma, RPN2106). Antibodies were used at the following concentrations: Anti-ATG5 (Abcam, ab108327; 1:1000), anti-LC3 (Abcam, ab192890; 1:1000), anti-ACTIN (Santa Cruz Biotechnology, I-19; 1:5000 [no longer commercially available]), anti-P53 (Cell Signalling Technologies, Clone 1C12; 1:1000), anti-P21 (Santa Cruz, SC-6246; 1:1000), anti-Histone H3 (Abcam, ab1791; 1:5000), anti-P16 (Santa Cruz, SC-1207; 1:1000), anti-HMGA1 (Abcam, ab129153; 1:1000), anti-NBR1 (Abcam, ab55474; 1:1000).

Western blot was performed according to the standard protocol. Proteins were extracted from cells and tissues with RIPA buffer (Beyotime, P0013B) complemented with protease inhibitors (Beyotime, P1005). Protein extracts were subjected to electrophoresis on 5× SDS-PAGE and transferred onto PVDF membranes (Immobilon: Millipore, IPVH00010). Immunoblots were blocked with 5% milk solution in TBS-Tween containing 0.1% Tween 20 and incubated with Anti-RCC2 antibodies (Abcamab154705 1:2000) or Anti-GADPH antibodies (Abcam ab181602 1:5000) overnight at 4℃. Subsequently, an appropriate HRP-conjugated secondary antibody (Beyotime A0216 1:5000) was added followed by incubation at room temperature for 1h. Western blots were visualized with chemiluminescence reagents (Sigma, RPN2106).