Anti p21waf1 cip1

For protein preparation, tissues and cells were homogenized in ice-cold HEN buffer (5 mM EDTA, 250 mM NaCl, 50 mM Hepes pH 7.3, 5 mM DTT) containing 1 mM Na₃VO₄, 0.2% IGEPAL CA-630 (Sigma) and 1% protease inhibitor cocktail (Sigma). Homogenized samples were centrifuged for 30 min. at high speed in a microcentrifuge, after which protein containing supernatant was collected. Protein concentration was determined using a protein assay kit from Bio-Rad. From each sample, 50 µg of protein was separated by electrophoresis on SDS–polyacrylamide gels and electroblotted onto PVDF membranes. Blots were incubated in blocking solution (5% non-fat milk in PBS, 0.1% Tween-20 [PBS-T]), followed by overnight incubation with the following antibodies: anti-HIF1α (Cayman, CAY-1006421; dilution 1∶500); anti-Glut1 (Millipore, 07-1401; dilution 1∶800); anti-p21^WAF1/Cip1 (Santa Cruz Biotechnology Inc., sc397; dilution 1∶200); and anti-β-actin (Abcam, ab6276; dilution 1∶5000). The membranes were then washed with PBS-T and incubated with either a HRP conjugated goat anti-rabbit IgG antibody (Thermo, 31460; dilution 1∶10000) or HRP conjugated sheep anti-mouse IgG antibody (ECL, NA931V; dilution 1∶10000). Antibody detection was performed with an enhanced chemiluminescence reaction (Clarity western ECL substrate; Bio-Rad).

The following reagents and chemicals were purchased from the indicated suppliers: anti-β-actin antibody and BITC from Sigma (St. Louis, MO, USA); horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG from Amersham (Arlington Heights, IL, USA); creatinine assay kit from Bio Vision (Milpitas, CA, USA); ALT and AST assay kits from Thermo Fisher Scientific Inc. (Middletown, VA, USA). Antibodies (anti-p21^WAF1/CIP1, anti-CDK2, anti-CDK4, anti-cyclin A, and anti-cyclin D1) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Standard western immunoblotting procedures were used with the following antibodies: anti-H3K36me1 (Abcam), anti-H3K36me2 (Abcam), anti-H3K36me3 (Abcam), anti-SETD2 (Santa Cruz Biotechnology), anti-hMSH6 (Santa Cruz Biotechnology), anti-histone 3.3, anti-histone 3, anti-SKP2 (Santa Cruz Biotechnology), anti-biotin (Santa Cruz Biotechnology), anti-Ub (Santa Cruz Biotechnology), anti-P300 (Abcam), anti-RNA Pol II (Abcam), anti-CyclinE (Santa Cruz Biotechnology), anti-CDK4 (Santa Cruz Biotechnology), anti-CyclinD1 (Abcam), anti-PCNA (Abcam), anti-ppRB (Abcam), anti-E2F1 (Abcam), anti-P18 (Abcam), anti-P21/WAF1/Cip1 (Santa Cruz Biotechnology), anti-PKM2, anti-c-Myc (Santa Cruz Biotechnology), anti-Chk1 (Abcam), anti-P62 (Abcam), anti-KDM4A (Abcam), and anti-β-actin (Abcam).
The primers were as follows: P62: P1, 5′-GCAGTATCCCAAGTTCAATT-3′; P2, 5′-TGGGAACAGGTGGTGGAGGA-3′; P62 promoter: P1, 5′-GATCATTCACACCTGTGGAC-3′; P2, 5′-GGACGAGTGGTCACCCTCTG-3′; pre-miR-675: P1, 5′-CCCAGGGTCTGGTGCGGAGA-3′; P2, 5′-CCCAGGGGCTGAGCGGTGAG-3; mature mi675: P1, 5′-TGGTGCGGAGAGGGCCACAGUG-3′; U6 primer: P1, 5′-GCTTCGGCAGCACATATACT-3′; P2, 5′-GGAACGCTTCACGAATTTGC-3′; and β-actin: P1, 5′-CTTCCTTCCTGGGCATGGAG-3′; P2, 5′-TGGAGGGGCCGGACTGGTCA-3′.