2.8 Immunoprecipitation
A549 cells were treated with 800 µM PQ with or without 5 mM lysine for 0, 15, or 30 min and lysed in RIPA buffer (Beyotime, P0013D) with addition of proteasome inhibitor (Roche, 04693124001). The lysates were quantified by Bicinchoninic acid (BCA) method and incubated with STIM1 antibody (1 µl/sample) overnight, followed by incubation with 20 µl protein A/G beads (Santa Cruz, SC-2003) for another 3h at 4°C. The immune complexes were washed three times with PBS and subjected to Western Blot. Specific antibodies were used for STIM1 (Cell Signaling Technology, 5668, 1:1000), ORAI1 (Santa Cruz, sc-377281, 1:500), and TRPC1 (Proteintech, 19482, 1:1000).