Ab28508

Cells were harvested and lysed in 1X RIPA lysis buffer (Thermofisher) containing 10 mM NaF, 1 mM Na₃VO₄, and protease inhibitor cocktail (Sigma). After centrifugation (14,000 × g for 10 min at 4 °C), supernatants were collected, and protein concentrations were determined with a DC protein assay (Bio-Rad). Proteins (10 μg) were separated by SDS-PAGE (12% gel) and transferred to PVDF membranes accordingly too Iblot2 methods (Invitrogen). The PVDF membranes were incubated overnight at 4 °C with antibodies against GAPDH (Abcam, Ab181603), TRPML1 (Abcam, Ab28508), BK channels (Abcam, Ab3586), and HIV-1 Tat (Santa Cruz, Sc-65916). The blots were developed with enhanced chemiluminescence, and bands were visualized and analyzed by our LI-COR Odyssey Fc Imaging System.

In U87MG cells, expression levels of TRPML1 and BK channels were knocked down using 1 μg/ml shRNA (Santa Cruz) against TRPML1 (Sc-44519) and BK channels (Sc-42511); control shRNA (Sc-108060) was used as a control. Jet prime reagent (2 μl) was used for transfection. Following 36 h incubation, 25 μg/ml puromycin (Invitrogen) was added to activate the shRNA promoter. After incubation for 2 to 3 days, cells were passaged to remove the dead and dying cells, and the remaining living cells were cultured for an additional 36 h prior to being taken for experimentation. Knockdown efficiency was confirmed by immunoblotting using antibodies against TRPML-1 (Abcam, Ab28508) and BK-channel (Abcam, Ab3586).

Total fibroblast cell lysates were subjected to whole protein quantification, separated on 4–12% precast gels (Lonza) by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and semi-dry transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences). The membranes were blocked in 5% non-fat milk (Bio Rad) in TBS-T (150 mM NaCl, 30 mM Tris base, pH 7.4, 0.1% Tween 20) for 1 h and immunoblotted using primary antibodies (1:1,000 dilution) against CLPP (Abcam, ab56455), MCOLN1 (Abcam, ab28508), MT-ND5 (Abcam, ab92624), NDUFA13 (Abcam, ab110240), NDUFB3 (Abcam, ab55526), NDUFB8 (Abcam, ab110242), TIMMDC1 (Abcam, ab171978) and UQCRC2 (Abcam, ab14745) for 1 h at RT or ON at 4 °C. Signals were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson Immuno Research Laboratories, Code: 111-036-045 and Code: 115-036-062, respectively, 1:5,000 dilution) for 1 h and visualized using ECL (GE Healthcare Life Sciences).