Anti cdkn1a p21

Adjusted protein lysates were supplemented with loading buffer (12% glycerol, 60 mM Na₂EDTA pH 8, 0.6% SDS, 0.003% bromphenol blue) and denatured for 10 minutes at 95 °C before SDS-PAGE. Proteins were wet-transferred to a nitrocellulose membrane at 80 V for 2 hours in transfer buffer (25 mM Tris-HCl pH 7.6, 192 mM glycine, 20% methanol, 0.03% SDS). Membranes were blocked in 5% milk powder diluted in TBST (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween 20), before an overnight incubation at 4 °C with the primary antibodies. After three times washing with TBST, membranes were incubated with HRP-coupled secondary antibodies for 1 hour at room temperature, followed by another three washes with TBST. Blots were developed with Pierce ECL Substrate Kit following the manufacturer’s instructions. The following antibodies were used: anti-FLAG M2 (mouse, F1804, Sigma-Aldrich), anti-GAPDH (mouse, ACR001P, Acris), anti-TRIM71 (rabbit, kind gift from the Yamanaka lab), anti-TRIM71 (rabbit, PAB19293, Abnova), anti-SHCBP1 (rabbit, 12672–1-AP, Proteintech), anti-Cdkn1a/p21 (rabbit, 2947, Cell Signaling Technology), anti-EGR1 (rabbit, 4154, Cell Signaling Technology), anti-UPF1 (rabbit, 9435, Cell Signaling Technology), anti-VINCULIN (mouse, V9131, Sigma-Aldrich), anti-ubiquitin (rabbit, 3933, Cell Signaling Technology) and anti-tubulin (mouse, T9026, Sigma-Aldrich).