Flag antibody

Precooled lysis buffer and PMSF (Thermo Scientific, #36978, 100:1) were used to lyse SW480 and HCT116 cells for 10 min. The supernatant was incubated with 2 µg of EFTUD2 antibody (Proteintech, #10208-1-AP), c-MYC antibody (Proteintech, #10,828-1-AP), Flag antibody (Abcam, #ab236777), and V5 antibody (Abcam, #ab309485) for 1 h. Following this incubation, 40 µL of protein A/G PLUS-Agarose (Santa Cruz, #20423) beads were added. After an overnight incubation at 4 °C, the beads were washed four times with immunoprecipitation buffer. Finally, the precipitate was dissolved in 40 µL of 1× electrophoresis sample buffer, boiled for 10 min, and subsequently subjected to Western blotting using specific antibodies.

Transfected HEK293AD cells fixed (HOW) on 18 mm glass coverslips were placed in 12-well Corning plates (Corning, Durham, NC, USA; catalog #3513) and washed with PBS (Sigma-Aldrich; catalog # D8537), then incubated for 10 min at room temperature with PBS + 0.25% Triton X100 (Research Products International, Mt. Prospect, IL, USA; catalog # 111,036). Coverslips were blocked in normal goat serum (Vector Laboratories, Newark, CA, USA; catalog # S-1000) for 1 h at room temperature, then incubated with one of the 7TM primary antibodies (1:500) and FLAG antibody (1:500, Abcam, Cambridge, UK; catalog # 117,495) overnight at 4 °C. The following day, coverslips were washed 3X with PBS and incubated with AlexaFluor488 goat anti-rabbit antibody (1:1000, Invitrogen, Waltham, MA, USA; catalog # A11008) for 1 h at room temperature. Coverslips were washed 3X with PBS and mounted on glass slides with ProLong Gold with DAPI (Vector Laboratories; catalog # H-1500). Slides were dried and imaged on a Nikon Eclipse Ti2-E Inverted Motorized Research Microscope with Perfect Focus System 4 and NIS-Elements AR Software (AI algorithms for optimal signal/noise imaging; 2D/3D deconvolution). Images shown are at 40 × magnification with exposure time of 50 ms for fluorescein isothiocyanate (FITC) and 400 ms for tetramethylrhodamine (TRITC) fluorophore channels.

For ChIP sequencing, chondrocytes were transfected with Lenti-3 × Flag-Runx1 (Lenti-3 × Flag-GFP as control). Coimmunoprecipitation was performed by using the Pierce^TM Agarose ChIP Kit (Lot#: TA265476). Runx1 overexpression was first examined by western blotting to confirm the success of transfection (Runx1 antibody and FLAG antibody were both verified). The antibody used for ChIP sequencing was the FLAG antibody (ab125243, Abcam). After immunoprecipitation, the DNA fragments were sequenced and identified at Shanghai Lifegenes Biotechnology (Shanghai, China).