Alexa fluor 594conjugated goat anti mouse igg h l

DSPP is not expressed in the HEK293 cells, so we choose this cell line to explore the location of the DSPP mutant. HEK293 cells were cultured in 24-well plates with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco^®, USA), 100 unit/mL penicillin, and 100 μg/mL streptomycin. The cells were transfected with DSP-pEGFP-N1-WT or DSP-pEGFP-N1-MUT using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). After 36 h of transfection, protein localization was detected by immunocytochemistry (ICC). The slides were incubated overnight at 4 °C with rabbit polyclonal anti-calnexin (CANX) antibody (cat: A0692, ABClonal, Wuhan, China) or mouse polyclonal anti-Golgi matrix protein (GM130) antibody (cat: ab169276, Abcam, Cambridge, UK) at a dilution of 1:600, followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG(H+L) (cat: AS039, ABClonal, Wuhan, China) or Alexa Fluor 594-conjugated goat anti-mouse IgG(H+L) (cat:A-11005, Invitrogen, USA) for 2 h. Subsequently, the cells were incubated for 2 h with 4',6-diamidino-2-phenylindole (DAPI). Cells were imaged using a confocal microscope (Zeiss LSM 800, Jena, Germany).