Amersham imagequant 800 system

Protein samples were mixed with an equal amount of 2× Laemmli reducing buffer and boiled for 10 min before being subjected to SDS-PAGE analysis on 12% polyacrylamide gels. Proteins were stained with InstantBlue solution (Expedeon) for visualization or were transferred onto PVDF membranes using iBlot Dry Blotting System (Thermo Fisher Scientific) at 20 V for 7 min. Following protein transfer, the PVDF membranes were immediately blocked with 1× casein blocking buffer (Sigma-Aldrich) and were probed with either horseradish peroxidase (HRP)-conjugated primary antibodies against His tag (Qiagen) or with rabbit primary antibodies against HA-tag (Sigma-Aldrich) followed by anti-rabbit HRP-conjugated secondary antibodies. Membranes were thoroughly rinsed with PBS containing 0.1% Tween 20 between the incubations. Chemiluminescent signals were detected using Amersham ImageQuant 800 System (GE Healthcare) after a brief incubation of the PVDF membranes with Immobilon Forte Western Chemiluminescent HRP Substrates (EMD Millipore). For reblotting with a different antibody after image acquisition, the membranes were incubated with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) for 30 min at room temperature, rinsed thoroughly before being blocked with casein blocking buffer, and probed with antibodies as described above.

The RIPA protein extraction method (Beyotime, Shanghai, China) was used to extract total protein from glioblastoma cells. Following centrifugation, the protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). For protein separation, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was employed, and PVDF membrane was utilized for protein transfer. After incubating the membrane overnight at 4°C with the specified antibodies, it was washed three times with TBST. Subsequently, the membrane was incubated with LAPTM4A-conjugated secondary antibodies at room temperature for the next hour. Finally, protein expression was detected using the Amersham™ Image Quant 800 system (GE Healthcare, Chicago, IL, USA).

Cytosolic fractionations for detection AIF and Cytochrome c were prepared in digitonin extraction buffer (0.015% digitonin, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl₂, 100 mM NaCl, 5 mM EDTA) supplemented with 1 mM phenylmetnylsulfonyl fluoride (PMSF). The whole-cell lysates were prepared in RIPA buffer (1× PBS, 0.5% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate (SDS)) containing 10 mM NaF, 10 mM β-glycerophosphate, 1 mM PMSF and protease inhibitor cocktail (Roche, Indianapolis, IN). Protein concentrations were detected with the Pierce BCA Protein Assay Kit (#23222, Thermo Scientific) according to the manufacturer’s instructions. Thirty μg of total proteins were separated by using SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After incubation with 5% dried skimmed milk, the NC membranes were incubated with the indicated primary antibodies at 4 °C overnight and then treated with the corresponding HRP-linked secondary antibodies at room temperature for 2 h. The protein bands were developed by using enhanced chemiluminescence (ECL) detection reagent (Beyotime, Shanghai, China) and then scanned using the Amersham ImageQuant 800 system (GE Healthcare, Piscataway, USA).