Sc 803

The purified recombinant proteins (4 µg/lane) were subjected to SDS-PAGE using 10% gels and then transferred to polyvinylidene difluoride membranes. Non-specific binding was blocked for 1 h with 2% bovine serum albumin (BSA) in PBS (pH 7.4) containing 0.1% Tween 20 at 25 °C. The membranes were incubated overnight in a solution containing anti-His antibody (Santa Cruz Biotechnology, sc-803) or anti-c-myc antibody (Santa Cruz Biotechnology, sc-40). The bands were visualized with ECL western blot detection kit (GE Healthcare)^{35 (link)}.

Next, 0.5–1.0 μg of each protein preparation was spotted onto a dry polyvinyl difluoride membrane (Pierce PVDF Transfer Membrane) and dried. The membranes were blocked with 5% nonfat dry milk in 1× Tris-buffered saline containing Tween 20 (TBST; 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.005% of Tween 20) for 1 h at room temperature. After that, the membranes were incubated in blocking buffer containing the anti-RHT-D1A (1:10,000 dilution in the blocking solution with 0.1% of Tween 20) or anti-Ape1L polyclonal antibody (1:5000 dilution in the blocking solution with 0.1% of Tween 20) on a rocker overnight at 4 °C. The membranes were then washed thrice in 1× TBST and incubated with a goat anti-rabbit antibody (ab6702, Abcam, Cambridge, UK) at a 1:20,000 dilution on a rocker for 1 h at room temperature. Next, each membrane was washed five times in 10 mL of 1× TBST for 5 min each time. A working substrate solution was prepared by mixing equal volumes of a H₂O₂ solution and luminal/enhancer solution. Each membrane was incubated in the working solution for 2 min in darkness, and Kodak X-Omat was exposed to the membrane. The 6xHis-tagged RHT-D1A protein was also detected with the anti-His antibody raised in rabbits (1:1000 dilution; sc-803; Santa Cruz Biotechnology) and an anti-rabbit Ig horseradish peroxidase–conjugated antibody (1:10,000 dilution).

In vitro GST pull-down assays were performed as we previously described (65 (link)). In brief, E. coli (BL21 DE3 strain) transformed with GST-TRIM21 or His-mutp53 (R175H) vectors was induced with 0.4 mM IPTG for 16 hours at 16°C to express GST-TRIM21 or His-mutp53 (R175H) proteins. The purified GST-TRIM21 proteins were immobilized on Glutathione-Sepharose beads (GE Healthcare, 17-0756-01), which were then incubated with purified His-mutp53 (R175H) or His-wtp53 proteins. GST protein alone was used as a negative control. After washing, proteins bound to the beads were analyzed by Western blot assays using an anti-His (Santa Cruz Biotechnology, sc-803) or anti-GST antibody (Santa Cruz Biotechnology, sc-138).