Ab56441

Cells for protein extraction were lysed with RIPA buffer and extracts were sonicated and centrifuged. Protein extracts were quantified with BCA kit (23,225, Thermo Fisher Scientific) according to the manufacturer’s protocol. 20 μg protein of samples was loaded in 12% polyacrylamide gels electrophoresis (PAGE). Electrophoresis was conducted at a constant amperage (30 mA) for approximately 2 h followed by the protein transference to a PVDF membrane at constant voltage (100 mV) for 1.5 h. 5% non-fat milk was used to block unspecific unions, and the primary antibodies mouse monoclonal anti-V-ATPase subunit V₀D₁ (ab56441, Abcam, used at 2.5 μg/mL) and monoclonal anti β-actin (a5316, SigmaAldrich, used at 1/20000), were incubated overnight. The next day, the secondary antibody was incubated for 1 h (goat anti-mouse IgG: 31430, Thermo Fisher Scientific, used at 1/10000). Finally, the membrane was developed using the chemiluminescence kit of BioRad according to the manufacturer’s protocol, and membrane chemiluminescence was detected with a Chemidoc MP Image System (BioRad). Image lab software (BioRad) was used for the quantification of bands.

Antibodies used were rabbit polyclonal anti-ATG16L1 (8090; Cell Signaling Technology, (Western blotting [WB] 1:1,000), rabbit polyclonal anti-ATG5 (2630; Cell Signaling Technology, WB 1:1,000), rabbit polyclonal anti-LC3A/B (4108; Cell Signaling Technology, WB 1:1,000, immunofluorescence [IF] 1:100), rabbit monoclonal anti-ATP6V1A (ab199326; Abcam, WB 1:2,000, IF 1:250), mouse anti-ATP6V1B1/2 (sc55544; SCBT, WB 1:1,000), mouse monoclonal anti-ATP6V0d1 (ab56441; Abcam, WB 1:1,000), mouse monoclonal anti-LAMP1 (555798; BD Biosciences, IF 1:100), mouse monoclonal anti-mCherry (ab125096; Abcam, WB 1:1,000), mouse monoclonal anti-GAPDH (ab8245; Abcam, WB 1:1,000), Alexa Fluor 488 polyclonal goat anti-rabbit IgG (A-11034; Thermo Fisher Scientific, IF 1:500), Alexa Fluor 568 polyclonal goat anti-mouse IgG (A-11004; Thermo Fisher Scientific, IF 1:500), Alexa Fluor 568 polyclonal goat anti-rabbit IgG (A-11011; Thermo Fisher Scientific, IF 1:500), HRP-conjugated anti-rabbit IgG (7074; Cell Signaling Technology, WB 1:2,000), and HRP-conjugated anti-mouse IgG (7076; Cell Signaling Technology, WB 1:2,000).