Stain free polyacryl amide gels

Western blot analyses were performed to analyze the expression of the BCL-2 family protein members and the effects of GX15-070 on LC3 cleavage. Total protein (30 μg) from untreated or vehicle-treated and GX15-070-treated cells (see above) were denatured by boiling in SDS sample buffer for 5 min. Proteins were separated by means of SDS-PAGE on stain-free polyacryl amide gels (Bio-Rad Laboratories) to enable loading control. After electrophoresis, optical densities of stained proteins in each lane were documented and verified with a CCD camera system and the Quantity One® software, respectively (both Bio-Rad Laboratories). When integrated optical densities of proteins in each lane did not differ more than maximal 10 %, proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories). After blocking with BSA, the blots were incubated overnight at 4 °C with the appropriate primary antibody (Cell Signaling Technologies) in TBS buffer containing 0.1 % Triton X100. After washing, an appropriate secondary antibody coupled to horseradish peroxidase was added. Detection of bound antigens was performed with an enhanced chemiluminescence detection kit (Amersham ECL Advance, GE Healthcare, Piscataway, NJ, USA). Using a CCD-camera system (Bio-Rad Laboratories), the signal intensities were evaluated.

The effects of incubation with ABT-737 or with obatoclax as the positive control on microtubule-associated protein 1A/1B-light chain 3 (LC3B) cleavage as a marker of autophagic cell death was analysed by western blot. 30 μg of total protein from treated and vehicle-treated cells (see above) were denatured by boiling for 5 min in SDS sample buffer. Proteins were separated by SDS-PAGE on stain-free polyacryl amide gels (Bio-Rad Laboratories) to enable loading control [80 (link), 81 (link)]. After electrophoresis, optical densities of stained total proteins in each lane were documented with a CCD camera system and verified using the Quantity One software (both Bio-Rad Laboratories). When the integrated optical densities of proteins in each lane did not differ more than 10 %, proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The blots were blocked with BSA and incubated with the LC3B primary antibody (Cell Signaling Technologies) in TBS containing 0.1 % Triton X100 overnight at 4 °C. An appropriate secondary antibody coupled to horseradish peroxidase was added and detection of bound antigens was performed by an enhanced chemiluminescence detection kit (Amersham ECL Advance, GE Healthcare, Piscataway, NJ, USA). Signal intensity was evaluated with a CCD-camera (Bio-Rad Laboratories).