Anti actin sc 1616

Nuclear protein extract were prepared as described previously [31] (link). Briefly, neonatal mice were euthanized by decapitation, and retinas were isolated immediately in pre-chilled PBS, which was frequently replaced with additional aliquots of PBS precooled on ice. Nuclear protein extracts were prepared by homogenizing retinas in ice cold nuclear extraction buffer (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM deferoxamine (Sigma), 0.1% NP-40, and 1× complete protease inhibitor cocktail (Roche)). Nuclei were collected by centrifugation, and resuspended in ice cold re-suspension buffer containing 20 mM HEPES-KOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.2 mM deferoxamine, 1× protease inhibitor cocktail, 0.2 mM phenylmethylsulfonyl fluoride, and 25% glycerol. The following antibodies were used for Western blotting: anti-HIF-1α (NB100-449, Novus Biologicals), anti-HIF-2α (NB100-132, Novus Biologicals), and anti-ß-actin (sc-1616; Santa Cruz Biotechnology).

Western blot membranes were incubated with the following primary antibodies: anti-ß-actin (sc-1616, 1 : 1000, Santa Cruz) and anti-ATF4 (sc-200x, 1 : 5000, Santa Cruz) and secondary antibody anti-goat (1 : 5000, Santa Cruz) and anti-rabbit (1 : 1000, Dako). Complete western blots are shown in Supplemental Figure S2.

U251-MG cells were seeded in 6-well plates at a density of 2 × 10⁵ cells/well and treated with AXT at a concentration of 0, 4, 8, 10 or 20 µM for 24 h in serum-free media. Cells were washed once with DPBS before lysis in RIPA buffer (Merck-Millipore). The total protein concentration was determined using the DC protein assay (Bio-Rad) and was quantified by measuring absorbance at 750 nm using a microplate reader (Bio-Tek), according to the manufacturer’s instructions. Ten µg of total protein from each sample was fractioned by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Following blocking for 1 h in 5% non-fat milk at room temperature (RT), the membranes were incubated with specific antibodies for 16 h at 4°C. Anti-p53 antibody (#OP03-100UGCN) was purchased from Merck-Millipore, anti-cyclin dependent kinase (Cdk) 2 (#sc-163), anti-phospho-Cdk2/3 (p-Cdk2/3) (#sc-12914), and anti-actin (#sc-1616) antibodies were purchased from Santa Cruz (TX, USA). After incubation with the primary antibodies at recommended dilution in 5% non-fat milk, the membranes incubated with HRP-conjugated secondary antibodies at RT for 2 h. The bands were detected using an enhanced chemiluminescence kit (Bio-Rad). The band intensities were measured using the ImageJ software [28 (link)].