Mouse control igg

The interacting plasmids were co-transferred into BmNS cells which were collected and lysed after transfection for 72 h. The 50 µL protein A + G Dynabeads (Life, Rockville, MD, USA) were incubated with anti-Flag/HA-tags antibody (Sigma, Rockville, MD, USA) or mouse control IgG (Beyotime, Shanghai, China) in 200 µL PBS (Phosphate Buffer Saline) for 30 min at 4 °C. Next, the lysed protein samples were added to the mixed systems for 1 h at 4 °C, after washing the Dynabeads two times with PBS. Finally, a 5 × sample loading buffer was added to the mixed systems, which were then boiled for 10 min to prepare the samples for the SDS-PAGE experiment. Following this, the protein bands were transferred onto a hydrophilic polyvinylidene fluoride (PVDF) membrane (Yeasen, Shanghai, China). The PVDF was incubated with primary antibodies and secondary antibodies (Beyotime, Shanghai, China), and visualized using a Western ECL Substrate (Bio-Rad, Hercules, CA, USA).

ChIP was performed using a ChIP assay kit (Beyotime, Haimen, China) according to the manufacturer's protocol. Chromatin solutions were sonicated and incubated with the anti-c-Jun antibody (Abcam, Cambridge, MA, USA) or mouse control IgG (Beyotime, Haimen, China) while rotating overnight at 4°C. The solution was washed according to the manufacturer's instruction. DNA-protein cross-links were reversed, and chromatin DNA was purified and subjected to PCR analysis using the Easy-Load PCR Master mix (Beyotime, Haimen, China). Primers 5’-TGTACGCGTGAGGCACATGGCAAAGG-3’ (forward) and 5’-GTTCTCGAGAGTGGGGAGGAAGTGGT-3’ (reverse) were used to amplify the β3GnT8 promoter sequence [19 (link)]. Following amplification, PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.

The samples were mixed with 5× SDS loading buffer and separated by 12% SDS–polyacrylamide gel, and then transferred onto a nitrocellulose membrane (Millipore, USA). The membrane was blocked with QuickBlock Western (Beyotime, China) and further incubated with appropriate primary antibodies at 4°C overnight. After being washed three times with TBST (Tris-buffered saline plus Tween 20), the membrane was incubated with a secondary antibody for subsequent detection by ECL enhanced chemiluminescence substrate (Thermo Scientific, USA). Flag, His, HA, and histone H3 antibodies, Cy3-labeled goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG secondary antibodies, and mouse control IgG were purchased from Beyotime. Goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and antiubiquitin (anti-Ub) were purchased from Abcam and Sigma, respectively. Tubulin, p53, HUWE1, and TRAF6 antibodies were prepared in our lab.