Chemiluminescent peroxidase substrate kit

Whole cell lysates were separated as previously described [36 (link)] on 10% or 15% SDS-polyacrilamide electroforesis gel respectively for Beclin 1 (Cell Signaling, Danvers, MA, USA) used at a final concentration of 1:1000, LC3 (Novus, Cambridge, UK) used at a final concentration of 1:2000, and BNIP 3 (Santa Cruz Biotechnology, Santa Cruz, TX, USA) used at a final concentration of 1:500. Samples were heat denatured for 5 min, loaded on standard Tris-HCl polyacrylamide gel and run on ice at 40 V for the stacking gel and 80 V for the running gel. Proteins were transferred onto a previously activated PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were then placed in TBS-T and 5% albumin for 1 h and probed overnight with the specific antibody at 4 °C. At the end of incubation time, membranes were washed and incubated with anti-mouse IgG (Cell Signaling, MA, USA) peroxidase conjugated secondary antibody (1:10,000) for 1 h at room temperature. Membranes were stripped and incubated with anti-actin monoclonal antibody (Sigma-Aldrich) as a loading control. Signal was detected by autoradiography (Kodak Biomax, Sigma-Aldrich) using the chemiluminescent peroxidase substrate kit (Sigma-Aldrich) then quantified by densitometric analysis using Quantity One software (Bio-Rad).

Whole cell lysates were separated as previously described [6 (link)] on 12.5% SDS-polyacrylamide electrophoresis gel for PTEN. Samples were heat denatured for 5 min, loaded on standard Tris-HCl polyacrylamide gel, and run on ice at 40 V for the stacking gel and 80 V for the running gel. Proteins were transferred onto a previously activated PVDF membrane (Bio-Rad, Hercules). Membranes were then placed in TBS-T and 5% albumin for 1 h and probed overnight with the specific antibody at 4°C. At the end of incubation time, membranes were washed and incubated with anti-mouse IgG peroxidase conjugated secondary antibody (1 : 10000) for 1 h at room temperature. Membranes were stripped and incubated with anti-actin monoclonal antibody as a loading control. Signal was detected by autoradiography (Kodak Biomax) using the chemiluminescent peroxidase substrate kit (Sigma) and then quantified by densitometric analysis using quantity-one software (Bio-Rad).

Cell pellets were resuspended in lysis buffer [RIPA buffer: 10 mM Tris–HCl (pH 7.6), 160 mM NaCl, 1 mM EGTA, 1% deoxycholic acid, 1%Triton, and 0.1% SDS] with protease inhibitors, incubated on ice for 30 min and then centrifuged at 12,000 g for 30 min; supernatants were collected. Whole cell lysates were heat denatured for 5 min and separated on 10% SDS-PAGE gels, run on ice at 40 V (for the stacking gel) and 80 V (for the running gel). Proteins were transferred onto a previously activated PVDF membrane (Bio-Rad, Hercules). Membranes were then placed in TBS-T and 5% albumin for 1 h and probed overnight with the specific antibody at 4°C. At the end of incubation, membranes were washed and incubated with anti-mouse IgG peroxidase conjugated secondary antibody (1:10,000) for 1 h at room temperature. Membranes were stripped and incubated with anti-actin monoclonal antibody (Sigma) as a loading control. Signal was detected by autoradiography (Kodak Biomax) using the chemiluminescent peroxidase substrate kit (Sigma) and then quantified by densitometry (Bio-Rad).