V-ATPase assay

To purify V-ATPase, two 15 cm petri dishes with subconfluent HeLa cells were transfected with pCDNA3.1HA-ATP6V1A plasmid (5 µg/plate). Three days later, cells were scraped off in SCA buffer supplemented with 1× protease inhibitor cocktail and ruptured using a Dounce glass homogenizer until ∼90% of cells were permeable for trypan blue. Ruptured cells were centrifuged at 1,000× g for 10 min and the obtained supernatant was further centrifuged at 20,000× g for 20 min. The pellet was lysed in 450 µL TBS lysis buffer supplemented with 1× protease inhibitor cocktail, incubated on ice for 30 min, and centrifuged at 20,000× g for 1 min. After determining the protein concentration, the supernatant containing 150 µg protein was precleared by rotation with 12 µL magnetic bead-conjugated rabbit IgG for 1 h at 4 °C. The precleared supernatant aspirated on magnetic stand was then rotated with 20 µL magnetic bead-conjugated rabbit mAb against HA for 2 h at 4 °C and the supernatant was aspirated on magnetic stand. The beads were washed 3× 5 min in 500 µL of TBS lysis buffer and suspended in 50 µL of V-ATPase reaction buffer (40 mM HEPES, pH 7.5, 1 mM MgCl₂, 100 mM KCl, 0.25 mM Sucrose, 5 µg/mL Oligomycin, 1 mM DTT) supplemented with 1× protease inhibitor cocktail.

To measure the V-ATPase activity, 15 µL anti-HA beads (V-ATPase) were mixed with 4.5 µg ΔN-STAT3sfGFP or 4.5 µg sfGFP (negative control) and the volume was adjusted to 150 µL with V-ATPase reaction buffer. The reaction was started by adding 1 mM ATP (final concentration) and incubating the samples for 30 min at 30 °C. The samples and free phosphate standard dilutions were then transferred to 96-well plates (50 µL/well) and the reactions were stopped by adding 100 μL BIOMOL Green Reagent. Plates were incubated at 25 °C for 20–30 min before determining OD620nm in a Varioskan Flash Multimode Reader. The amount of ATP hydrolyzed was calculated using the standard curve created by the free phosphate dilutions analyzed on the same plate.