Horseradish peroxidase conjugated goat anti mouse and anti rabbit antibodies

Mouse monoclonal antibody (mAb) anti-Ago2 (MA2) was a gift from Dr. O'Carrol. mAb to p27 was from BD Transduction Laboratories (Franklin Lakes, NJ, USA), and rabbit polyclonal Ab to p38 (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mAbs to Nucleoporin 153 (QE5) and to Ahnak (EM-09) and rabbit polyclonal Ab to RBM24 (94567) were from Abcam (Cambridge, UK). mAb to Myosin Heavy Chain (MF20) was obtained from Dr. Fischman and mAb to myogenin (F5D) was a gift from G Cossu. TRITC-conjugated goat anti-mouse antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were from Santa Cruz Biotechnology.

Briefly, CB or PMF CD34+ was lysed in 50 mm Tris pH 7.4, 150 mm NaCl, 10 mm KCl, 1 mm EDTA, 20 mm NaF, 5 mm DTT, 0,25% Na deoxycholate, 0,1% Nonidet P‐40 and protease inhibitor (Complete, catalog #1697468, Roche, Milano, Italy). Total cell lysates (30 mg·mL⁻¹ for each sample) were resolved by electrophoresis on a 10% SDS/polyacrylamide gel and transferred to nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). The membranes were checked for loading and transfer efficiency by staining with Red Ponceau. Membranes were blocked with 5% BSA in 0,1% Tween‐20 for 1 h at room temperature (RT), washed, and incubated with primary antibodies: (a) mouse monoclonal anti‐SOD2 antibody (1 : 20 000 dilution, SOD‐2 (A‐2) catalog #sc‐133134, Santa Cruz Biotechnology, Inc., Heidelberg, Germany) overnight at 4 °C; (b) rabbit polyclonal anti‐actin antibody (1 : 2000 dilution, Thermo Fisher Scientific, Inc., Waltham, MA, USA, catalog #PA1‐16889) for 1 h at RT. Horseradish peroxidase‐conjugated goat anti‐mouse and anti‐rabbit antibodies (catalog #sc2005, #sc2004 Santa Cruz Biotechnology Inc) were added at 1 : 5000 and 1 : 10 000, respectively, for 1 h at RT. BM Chemiluminescence Blotting Substrate (POD; Roche) visualized proteins. imagej software was used to quantify the protein level.