Ab32400

The lysates generated from mice brain tissues or primary cortical mixed cells were precleared with Protein G beads and followed by being incubated with 5 μg/ml anti-ASK-1 antibody (8662; Cell Signaling Technology) or anti-MLL1 antibody (ab32400, Abcam) overnight at 4°C. Then, Protein G beads were added and incubated for 2 h at 4°C. After that, beads were washed using lysis buffer for three times and were directly boiled in 1× Laemmli buffer. The coprecipitated proteins were further separated and analyzed by SDS-PAGE and Western blotting analysis.

Immunostaining was performed on paraffin-embedded sections. After blocking with 5% bovine serum albumin (BSA), tissues were incubated with the following antibodies overnight at 4°C: antibodies directed against MLL1 (ab32400, Abcam) and cleaved caspase-3 (9661S; Cell Signaling Technology) were used to detect the target proteins. All slides were visualized using DAB substrate (Beyotime, China) and quantified by H score. The H score was calculated using the following formula: H score = Pi (i), where i is the intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%.

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysate buffer (Beyotime, China). The same amount of protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinyl difluoride (PVDF) membrane. After soaking in protein-free fast block buffer (Beyotime, China), the membrane was incubated overnight with one of the following antibodies at 4°C: anti-ASK-1 antibody (8662, Cell Signaling Technology), anti-pASK-1 antibody (3764, Cell Signaling Technology), anti-STRAP antibody (AP29336; One World Lab), anti-14-3-3 antibody (9636; Cell Signaling Technology), anticleaved caspase-3 antibody (ab2302, Abcam), anti-TNF-α antibody (ab1793, Abcam), anti-MLL1 antibody (ab32400, Abcam), anti-WDR5 antibody (ab178410, Abcam), and anti-tubulin antibody (ab6160, Abcam). Then, the secondary antibody was used to incubate the membrane at room temperature for 0.5 h. Finally, the membrane was observed by ECL Plus Kit (Beyotime, China) and quantified using the ImageJ program (National Institutes of Health, USA).