Nel105

Cell lysates were prepared from cell pellets and equal amounts of protein loaded in each lane of 7.5 or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes as previously described [18] (link). Phosphorylated and total proteins were detected sequentially on the same membrane using specific primary antibodies, appropriate secondary antibodies conjugated to HRP and enhanced chemiluminescence (NEL105, Perkin-Elmer). Bands were quantitated by densitometry (Molecular Dynamics) using CareStream MI SE software.

Cells (5 × 10⁵) were collected at various time points and then washed with ice-cold PBS twice before adding lysis buffer (M-PER Mammalian Protein Extraction reagent, Thermo Scientific, Cat No. 78501) and cocktail inhibitor (Sigma, 5 μg/ml, Cat No. P8340). Equal amounts of protein were loaded into each well and separated by NuPAGE 4–12% Bis-Tris gel, followed by transfer onto PVDF-membranes (Bio-Rad, Cat No. 162-0177). Membranes were blocked with 5% non-fat milk in PBS-T for 1 h at room temperature. The blots were then incubated with antibodies against GRPR, Caspase-3, p-AKT1, AKT1, p-AKT2, AKT2, p-mTOR, mTOR, p-MAPK, MAPK, p-RPS6, RPS6, p-p38 MAPK, p38 MAPK CD133, Sox2, and ALDH for 1 h at 4°C. Goat anti-rabbit IgG secondary (1:5000; Santa Cruz Biotechnology, Cat No. sc-2004) was then incubated for 45 min at room temperature. Immunoblots were developed by using the chemiluminescence detection system (PerkinElmer, Cat No. NEL105) and autoradiography was performed. β-actin was used as a loading control.

Cells (5X10⁵) were collected at various time points, and then washed with ice-cold PBS twice before adding lysis buffer (M-PER Mammalian Protein Extraction reagent, Thermo Scientific, 78501) and cocktail inhibitor (5 µg/ml, Sigma, P8340). Equal amounts of protein were loaded into each well and separated by NuPAGE 4-12% Bis-Tris gel, followed by transfer onto PVDF-membranes (Bio-Rad, 162-0177). Membranes were blocked with 5% non-fat milk in PBS-T for 1 h at room temperature (RT). The blots were then incubated with antibodies against LC3 I &II. for 1 h at 4°C. Goat anti-rabbit IgG secondary antibody (1:5000; Santa Cruz Biotechnology, sc-2004) was then incubated for 45 min at RT. Immunoblots were developed by using the chemiluminescence detection system (PerkinElmer, NEL105) and autoradiography was performed. β-actin was used as a loading control.