Pa5 22688

For immunofluorescence assays, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton. After blocking with 1% bovine serum albumin (BSA), cells were then incubated with the primary anti-α-actin antibody (ab5694, 1:500, abcam, USA), anti-calnexin antibody (AF18, 1:500, Thermo Fisher Scientific, USA), anti-Tom20 antibody (sc-17764, 1:500, Santa Cruz Biotechnology Inc., USA), anti-derlin (sc-390289, 1:500, Santa Cruz Biotechnology Inc., USA), or anti-green fluorescent protein (GFP) (PA5-22688, 1:1,000, Thermo Fisher Scientific, USA) for 60 min at 37°C, respectively, followed by secondary antibodies, Alexa 488 immunoglobulin G (IgG) (A32723, 1:1,000) or Alexa 568 IgG (A11011, 1:1,000) (Thermo Fisher Scientific, USA), in a dark chamber. Cells were examined using a ZEISS LSM510 META laser-scanning confocal microscope (Carl Zeiss, Germany). Fluorescence images were acquired by a Plan-Neofluar 20×/0.40 LD or Plan-Apochromat 63×/1.40 Oil objective with either 488 nm laser excitation [520 band pass (BP) emission] or 543 nm laser excitation [570 long pass (LP) emission].