Ab264168

The binding between KDM5C and PFDN5 promoter was assessed through the ChIP-qPCR assay. In short, CRC cells were crosslinked in 1% formaldehyde and then ultrasonicated on ice to generate DNA fragments. The resulting product was incubated with anti-KDM5C (1:50, ab264168, Abcam Inc., Cambridge, MA, USA), anti-H3K4me3 (1:50, ab8580, Abcam) or the control immunoglobulin G (IgG) at 4 ℃ overnight. Subsequently, the immunoprecipitated complexes were eluted and de-crosslinked with NaCl solution overnight. Chromatin DNA was recovered from the precipitate, dissolved in deionized water, and analyzed using qPCR.

Total protein from the cells was extracted using radio-immunoprecipitation assay cell lysis buffer (Cell Signaling Technology) supplemented with the protease inhibitor (Roche Ltd, Basel, Switzerland). The protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following a 1-h block in 5% non-fat milk at room temperature, the membranes were incubated with the primary antibodies against KDM5C (1:1,000, ab264168, Abcam), PFDN5 (1:1,000, ab129116, Abcam), ATG7 (1:1,000, ab52472, Abcam), P62 (1:1,000; NBP1-48320, Novus Biologicals, Littleton, CO, USA), Beclin1 (1:1,000, #3495, Cell Signaling Technology), LC3 (1:1,000; #3868, Cell Signaling Technology), Vimentin (1:1,000, NBP1-31327, Novus Biologicals), N-cadherin (1:1,000, NBP2-19457, Novus Biologicals), GAPDH (1:1,000; ab181602, Abcam) at 4 °C overnight. Subsequently, the membranes were incubated with the HRP-conjugated IgG (1:20,000, ab6721, Abcam) at room temperature for 2 h. Protein blots were developed using enhanced chemiluminescence reagent and analyzed using Image J.