Asp175 clone 5a1e

Cells were lysed with lysis buffer (20 mM Tris-HCl (pH8.0), 1% SDS, and 1 mM DTT). Blots were probed with a mouse monoclonal antibody against BNIP3 (Clone ANa40; Abcam; ab10433), rabbit monoclonal antibody against cleaved Caspase3 (Asp175; Clone 5A1E; Cell Signaling Technology; #9664), rabbit monoclonal antibody against SAPK/JNK (Clone 56G8; Cell Signaling Technology; #9258), rabbit monoclonal antibody against phospho SAPK/JNK (T183/Y185; Clone 81E11; Cell Signaling Technology; #4668), rabbit polyclonal antibody against phospho c-Jun (S63; Cell Signaling Technology; #9261), rabbit monoclonal antibody against p44/42 MAPK (Erk1/2; Clone 137F5; Cell Signaling Technology; #4695), rabbit monoclonal antibody against phospho p44/42 MAPK (T201/Y204) (Clone D13.14.4E) (Cell Signaling Technology; #4370), rabbit polyclonal antibody against LC3B (Cell Signaling Technology; #2775), rabbit polyclonal antibody against p62 (MBL; PM045), and mouse monoclonal antibody against Actin (Clone C4) (Merck-Millipore, Billerica, MA, USA; MAB1501). Horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG secondary antibody (Cell Signaling Technology; #7076, #7074) was used as a probe, and immunoreactive bands were visualized with the Immobilon Western Chemiluminescent HRP substrate (Merck-Millipore). The band intensity was measured using ImageJ software (NIH, Bethesda, MD, USA).

Samples were fixed in 4% paraformaldehyde, embedded in optimal cutting temperature (OCT) compound, frozen, and sectioned at 10 μm. Sections were then subjected to immunohistochemical analysis as previously described.^{40 (link)} Briefly, the sections were stained with rabbit monoclonal antibody against cleaved Caspase3 (Asp175; Clone 5A1E; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibody against GFP (ThermoFisher Scientific, Waltham, MA, USA), and mouse monoclonal antibody against LAMP2 (abcam, Cambridge, UK). For immunofluorescence analysis using the LC3 antibody (MBL, Nagoya, Japan), the cells were permeabilized with PBS containing 100 μg/ml Digitonin (Sigma-Aldrich, Saint Louis, MO, USA) instead of PBST (PBS, 0.5% Triton X-100). Images were obtained using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan) and were analyzed using BZ Analyzer Software (Keyence). For the quantitation of autophagosomes, images with at least 100 representative cells per condition in three independent experiments were analyzed using IN Cell Investigator software (GE Healthcare, Chicago, IL, USA).