Anti myc antibody

HEK293T cells (5 × 10⁴) expressing indicated STIM1 or Tmem178 mutants were plated on glass coverslips pre-coated with poly-lysine, fixed in 4% formaldehyde for 20 min and permeabilized with 0.1% Triton-X-100 in phosphate-buffered saline (PBS) for 6 min. Cells were then washed with PBS twice and blocked in 0.2% BSA at room temperature (RT) for 30 min prior to overnight incubation at 4 °C with the anti-HA antibody (Cell signal Technology, 3724, 1:1600, MA, USA), anti-Myc antibody (Biolegend, 906301, 1:1000, CA, USA), or the anti calnexin antibody (Santa Cruz, Sc-6465, 1:25, CA, USA). Cells were gently washed and secondary antibodies (Thermo Fisher Scientific, A11058 and A21206, 1:1000, NJ, USA) were added at RT for 1 h. Coverslips were mounted using VECTASHIELD anti-fade mounting medium with DAPI (Vector Laboratories, H-1200, Burlingame, CA, USA). Fluorescent signals were captured by using a Nikon Eclipse 80i microscope and a Nikon DS-Qi1MC camera (Nikon, CO, USA).