IP and silver staining

Protein lysates were collected 96 h post-transfection from Hs578T(KO) cells transfected with EV, Q5, Q5H, or Y150F on which confluency was reached; 1 mg of whole-cell protein lysate was incubated overnight at 4°C with Protein A/G beads that were cleaned twice with 1× Dulbecco-PBS (ThermoFisher) along with 10 μg of anti-human PANX1 antibody (PANX1 CT-412) cross-linked to Pierce Protein A/G-Agarose beads (Thermo Scientific). Subsequently, bound proteins underwent four washes with 500 μl of IP buffer. Beads were collected and resuspended in 2× Laemmli buffer with 10% β-mercaptoethanol, boiled for 5 min, and supernatant was collected after spinning down. Supernatant then was subjected to 10% SDS–PAGE and gel was stained with Pierce Silver Stain Kit (ThermoFisher Scientific) according to manufacturer’s protocol.