Ab236256

Cells were collected, lysed in lysate (phosphatase inhibitor, protease inhibitor and PMSF), and protein quantification was performed by BCA method (Thermo Fisher Scientific, China). 10-20 μg protein was loaded on 8%-12% 30% acrylamide-Bis gel, then transferred through 0.22 μm pore PVDF membrane (Merck Millipore, USA), and blocked with 5% nonfat milk powder for 1 h., respectively, add primary antibodies (Rabbit polyclonal to IFIT1, 1:1000, ab236256; Rabbit monoclonal [EPR10009] to PARM-1, 1:1000, ab168369, abcam) and incubate overnight at 4°C. The next day, secondary antibodies (Goat Anti-Rabbit actin, 1:2000, ab8226, abcam) were added and incubated at room temperature for 1 h, and then luminescent solution (Thermo Fisher Scientific, China) was added for exposure and color development. Image J software was used for analysis, and the relative protein content was expressed as the gray value of the corresponding protein band/the gray value of the actin protein band, which was repeated three times.

Protein extracts were prepared with RIPA Lysis buffer (Millipore) containing 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 500 mM NaCl, 1 mM EDTA, and 1 × protease inhibitor cocktail (Roche Applied Science). To detect LGALS3BP secreted protein, cell supernatant with trichloroacetic acid precipitation was prepared according to protocol [113 (link)]. The protein extracts were resolved by SDS-PAGE, transferred to a PVDF membrane, and incubated with the indicated antibodies. Detections were performed using ECL reagents. The antibodies used are as follows: custom-designed mouse monoclonal antibody against BORIS, mouse monoclonal antibody against CTCF (Santa Cruz, sc-271514), mouse monoclonal antibody against Tubulin (Abcam, ab7291), rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), Rabbit IgG (Abcam, ab37415), rabbit monoclonal against histone H3 (Abcam, ab201456), rabbit monoclonal against LINE-1 ORF1p (Abcam, ab216324), rabbit monoclonal against IRF7 (Thermo Fisher Scientific, SC0617), rabbit polyclonal against LGALS3BP (Thermo Fisher Scientific, PA5-79597), rabbit polyclonal against IFIT1 (Abcam, ab236256), rabbit polyclonal against IFI44 (Thermo Fisher Scientific, PA5-96967), rabbit polyclonal against Aim2 (Cell Signaling, #63660), rabbit polyclonal against OAS1 (MyBioSource, MBS129033).