Rabbit anti flag

Protein samples from both HEK293 cells and primary keratinocytes were lysed in RIPA buffer with protease/phosphatase inhibitor and denatured at 95 °C for 5 min or 50 °C for 10 min. Equal amounts of protein were loaded and electrophoresed on 8% SDS-polyacrylamide gels for 1.5 h at 120 V. The proteins were next transferred to a PVDF membrane in a cold room for 2 h at 120 V. After blocking for 1 h with BLOCK ACE (KAC, UKB80) at RT, the membrane was incubated with antibodies to detect target bands. The primary antibodies included mouse anti-FLAG (Sigma, F3165, 1:2000), rabbit anti-FLAG (Santa Cruz, sc-807, 1:1000), mouse anti-MYC (MBL, 1:1000), and HRP-conjugated anti-GAPDH (Cell Signaling, 3683, 1:1000). The secondary antibodies consisted of anti-mouse IgG (Cell Signaling, 7076, 1:10000) and anti-rabbit IgG (Cell Signaling, 7074, 1:10,000). All proteins were incubated with an ECL kit (Cytiva), and the blots were imaged using a LAS-3000 mini (Fujifilm). Quantification was conducted with ImageJ.

Twenty-four h after transfection, HEK293 cells were gently rinsed with cold 1× PBS and collected in 600 uL RIPA buffer (Thermo Scientific, 89900) supplemented with a protease inhibitor and a phosphatase inhibitor. Next, the cell lysate was placed on ice for 30 min with pipetting every 10 min followed by centrifugation at 12,000×g for 30 min. The following steps were all performed at 4 °C. Each supernatant sample was first cleared for 1 h with 1 μg of off-target monoclonal IgG1 antibody produced in mice then transferred into 50 μL of a pre-washed, Protein G Mag Sepharose bead (Cytiva, 28951379) slurry for 30 min of pre-cleaning. The cell lysates were next incubated with 2 μg of mouse anti-MYC (MBL, M047-3) or mouse anti-FLAG (Sigma, F3165) O/N, and the bead pellet was discarded. On the second day, the antibody-antigen complex was precipitated with 25 μL of fresh protein G beads for 3 h with subsequent washing in lysis buffer three times. The complex was then eluted from the beads in 40 μL of 1× sample buffer (Bio-Rad) by heating at 50 °C for 10 min. Five microlitres of each sample was loaded for Western blotting. Mouse anti-MYC and rabbit anti-FLAG (Santa Cruz Biotechnology, sc-807) antibodies were used to probe for TRPV3 and TMEM79 bands, respectively.