Anti kdm4b

Whole-cell lysates were obtained by resuspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Shao et al., 2019 (link); Weng et al., 2019 (link)). Specific antibodies or preimmune immunoglobulin G (IgG) (P.I.I.) was added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X sodium dodecyl sulfate (SDS) electrophoresis sample buffer. Western blot analyses were performed with anti-ADAM10 (Proteintech, 25900-1), anti-ADAM17 (Proteintech, 25209-1), anti-ADAM19 (Proteintech, 22216-1), anti-BRG1 (Abcam, ab110641), anti–β-catenin (Cell Signaling Tech, 8480), anti-KDM4A (Cell Signaling Tech, 5328), anti-KDM4B (Cell Signaling Tech, 8639), anti-KDM4C (Bethyl Laboratories, A300-885A), and anti–β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, and 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Chen et al., 2020c (link); Dong et al., 2020 (link); Lv et al., 2020 (link); Mao et al., 2020a (link),b (link); Wu et al., 2020b (link); Hong et al., 2021 (link)). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Western blot analyses were performed with anti-ERG1 (Cell Signaling Tech, 97249), anti-KDM4B (Cell Signaling Tech, 8,639), anti-TCL (Sigma, HPA003050), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.

Total cells and tissues were extracted using NP-40 lysis buffer in the presence of a protease inhibitor cocktail (KeyGEN, Nanjing, China). Proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Merck KGaA, Darmstadt, Germany). Membranes were probed with anti-KDM4B (8639; Cell Signaling Technology), anti-HAX1 (sc-166845; Santa Cruz Biotechnology), anti-cleaved caspase-9 (7237; Cell Signaling Technology), anti-cleaved caspase-3 (9664; Cell Signaling Technology), anti-histone H3 (ab1791; Abcam), anti-H3K9me3 (9754S; Cell Signaling Technology), anti-α-tubulin (12152; Cell Signaling Technology), anti-Bcl-2 (2870; Cell Signaling Technology), and anti-Bax (2772; Cell Signaling Technology) antibodies. Protein bands were incubated with HRP-conjugated antibodies and visualized using electrochemiluminescence by a chemiluminescence instrument.