Anti hsc70 mouse monoclonal antibody

Total cell extracts were prepared and western blotting was carried out as previously described.^{1 (link)} Blots were incubated with a primary polyclonal antibody and then with an appropriate peroxidase- conjugated secondary antibody (anti-rabbit and antimouse IgG horseradish peroxidase [HRP] antibodies from Cell Signaling, cat. n. 7074S, and 7076S, respectively) and anti-goat IgG HRP antibody from Southern Biotech (cat. n. 6160-05). Proteins were detected using a chemiluminescent system (Clarity Western ECL, cat. n. 170-5060, Biorad). Rabbit polyclonal antibodies were previously described for RINF,^{1 (link)} P85/PI3KR1,^{42 (link)} or commercially purchased for anti-RINF, anti-SMAD7 (cat. n. 16513-1-AP, cat. n. 25840-1-AP, ProteinTech), anti-b-actin (ACTB, cat. n. A1978, Sigma-Aldrich), anti-phospho-SMAD2/3 (cat. n. 8828, Cell Signaling), and anti-HSC70 mouse monoclonal antibody (SC-7298, Santa Cruz Biotechnology).

Protein extraction was performed using lysis buffer [10% glycerol, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 400 mM NaCl, 0.5% NP40, 4 μg/ml aprotonin, PMSF, proteasome inhibitor MG-132 and 1 mM DTT]. Total protein (10 μg) was electrophoresed on a 10% polyacrylamide gel, and then electroblotted at 300 mA for 90 min on a nitrocellulose membrane (Invitrogen, Carlsbad, CA, USA). Western blot analysis was used to confirm the protein expression of HIF-1α and HSC70: These proteins were detected using anti-HIF-1α rabbit polyclonal antibody (1:1,000) (Cell Signaling Technology, Cat. no. 3716) and anti-HSC70 mouse monoclonal antibody (1:1,000) (Santa Cruz Biotechnology, Cat. no. sc-7298). HSC70 expression was used as a loading control. The signals were detected using the ECL Select Western Blotting Detection System (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and Image Quant LAS 4000 software (GE Healthcare Life Sciences).