Mouse anti human β actin clone ac 15

Treated cells were lysed for 10 min on ice in suspension buffer (0.1 M NaCl, 0.01 M Tris-Cl (pH 7.6), 0.001 M EDTA (pH 8.0), 1 μg/ml leupeptin, 1 μg/ml aprotinin, 100 μg/ml PMSF). An equal volume of 2 X SDS gel loading buffer (100 mM Tris-Cl (pH 7.6), 4% (w/v) SDS, 20% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol 0.2% bromophenol blue) was immediately added to the cell suspension followed by sonication for 20 s with 1-s pulses (40/output, 200 W). The lysate was clarified by centrifugation at 12,000 x g for 10 minutes at room temperature followed by boiling at 95°C for 5 min. Approximately equal numbers of cell equivalents were loaded and SDS-PAGE was performed using 10% (v/v) resolving gels and 5% (v/v) stacking polyacrlyamide gels. Proteins were transferred to 0.45 μm nitrocellulose membranes. Membranes were blocked in TBS+5% milk for 1 h at room temperature, then probed with Mouse Anti-human-β-actin clone AC-15 (Sigma-Aldrich) in TBS+5% milk for 2 h at room temperature or overnight at 4°C. Membranes were washed and incubated with Goat anti-mouse IgG, Alkaline Phosphatase conjugated antibody (Promega) in TBS+5% milk for 1 h at room temperature. Membranes were washed and incubated in BCIP (Sigma-Aldrich) reagent for 5 min at room temperature and photographed.

Freshly isolated keratinocyte populations were harvested for CD271 in lysis buffer pH 7.5 (150 mM NaCl, 15 mMMgCl, 1 mM EGTA, 50 mM Hepes, 10% Glycerol, 1% Triton), or in RIPA buffer. WB was performed as previously described.^{9 (link)} Membranes were first incubated in blocking buffer and then overnight at 4 °C with primary antibodies: mouse anti-human CD271, clone ME20.4 (Upstate, Lake Placid, NY, USA), mouse anti-human β1 integrin, clone 12G10 (Abcam, Cambridge, UK), rabbit anti-FOXM1, clone D3F2B (Cell Signaling Tech, Danvers, MA, USA), rabbit anti-p63(ΔN) (BioLegend, San Diego, CA, USA), rabbit anti-Survivin (Boster, Pleasanton, CA, USA), rabbit anti-p16INK4 (Bethyl Laboratories Inc, Montgomery, TX, USA), rabbit anti-Ki67 (Abcam), mouse anti-human β-actin, clone AC-15 (Sigma). Then membranes were incubated with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Biorad, Hercules, CA). Membranes were developed in Clarity Max ECL substrate (BioRad) and images were captured with ChemiDoc Imaging System (Biorad). The band intensity was quantitatively determined using Fiji-ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA), and protein levels’ intensity was normalized to β-actin expression.