Ab36938

Total protein in tissues and cells was extracted with RIPA lysis buffer (R0010, Solarbio) containing PMSF. The cells were incubated for 30 min on ice, centrifuged at 12000 r/min, and 4°C for 10 min, and the supernatant was taken. The BCA kit (23225, Pierce) was employed to determine protein concentration of each sample. A 10% SDS-PAGE (P0012A, Beyotime Institute of Biotechnology, Shanghai, China) was adopted to dissolve proteins, which were transferred to a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) by wet transfer. The PVDF membrane was blocked with TBST buffer containing 5% skimmed milk powder for 2 h. The blots were probed with the primary antibody to rabbit KDM6A (1 : 1000, ab36938, Abcam, UK), NOTCH2 (1 : 1000, Ak098372, Abcam, UK), Abcb1a (1 : 1000, ab3373, Abcam, UK), IL-1β (1 : 1000, S328, NIBSC, UK), IL-6 (1 : 1000, Tyr705, Abcam, UK), IL-8 (1 : 1000, S333, Abcam, UK), TNF-α (1 : 1000, C432, PA, USA), Bcl-2 (1 : 1000, ab32124, Abcam, UK), Bax (1 : 1000, 6A7, Abcam, UK), caspase-3 (1 : 1000, H-277, Abcam, UK), and β-actin (1 : 1000, AC15, Abcam, UK). HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China, 1 : 5000) was then incubated with cells. The blots were developed with the ECL and quantified with Quantity One v4.6.2 software.

Cell lysates were prepared and processed as described [16 (link)]. Antibodies were used at the following dilutions: β-actin (MAB1501, 1:1,000; Chemicon), KDM6A (ab36938, 3μg/mL; Abcam), p21^CIP1 (ab109520, 1:750 and ab109199, 1:1,000; Abcam), FLAG (F-3165, 1:1,000; Sigma) and HRP-conjugated secondary anti-rabbit (1:10,000; Amersham) and anti-mouse (1:10,000; Amersham). Antigen/antibody complexes were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) and electronically acquired with a Kodak 4000R Image Station (Kodak) equipped with Carestream Molecular Imaging Software.

Cells were lysed by radioimmunoprecipitation assay lysis buffer in the presence of protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3 (P8340, P5726, and P0044, Sigma-Aldrich). Cell lysates were electrophoresed on 8%–12% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. Antibodies were against JMJD3, Oct4, UTX and H3K27me3 (ab38113, ab19857, ab36938, and ab6002; Abcam, Inc. Cambridge, MA), aldehyde dehydrogenase (ALDH), β-actin (sc-166362, sc-4778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and PHF20 (D96F6, Cell Signaling Technology, Inc., Danvers, MA). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected by enhanced chemiluminescence reagent (Millipore, Billerica, MA).