Investigating TRPC1 and PI3K interactions

The 6 × 10⁵ non-transfected or 1 × 10⁶ transfected PANC-1 cells grown in the three different pH conditions were seeded in 10 cm Petri dishes and collected after 72 h. As previously described [37 (link)], 500 µg of proteins were used for co-immunoprecipitation with SureBeads™ Protein A Magnetic Beads (Bio-Rad, France). Beads were washed thoroughly, according to the manufacturer’s protocol. Then, 1 µg of either TRPC1 antibody (Abcam, Waltham, MA, USA), PI3K p85α antibody (Bioworld Technology, tebu-bio, France), or a control HRP-linked anti-rabbit IgG antibody (Cell Signaling Tech., Danvers, MA, USA) were resuspended with the beads for 30 min. Protein lysates were subsequently washed and added to the beads, which were slowly rotated for 2 h at room temperature. After another sequential washing step of the beads, proteins were eluted according to the manufacturer’s protocol. After denaturation, proteins were subjected to Western blotting as described above. To detect the input, 50 µg of proteins from the corresponding co-immunoprecipitation samples were used.