HepG2 cells were fixed by 4% paraformaldehyde for 10 minutes at room temperature after being washed by PBS twice. Then cells were blocked and permeabilized with 0.1% Triton X-100 (MP Biomedicals) and 10% FBS diluted in PBS for 30 minutes at room temperature. After that, cells were incubated with primary antibody SREBP1 (Thermo Fisher Scientific, 1:100), SREBP2 (Abcam, 1:100), INSIG1 (Proteintech, 1:200), CALNEXIN (Enzo, 1:200), and PDI (MilliporeSigma, 1:200) incubated at 4°C overnight. After 5 washes with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) secondary antibody (Life Technologies) for 1 hour at room temperature with gentle shaking. At last, the samples were washed with PBS 3 times before staining the nucleus with DAPI for 5 minutes. Immunofluorescence images were obtained and analyzed with Zeiss 710 NLO confocal microscopy.
For fluorescence intensity quantification, ImageJ was applied, and relative intensity was quantified by intensity divided by view area. For colocalization, the rate was quantified using ImageJ with colocalization finder. For each sample, no less than 5 representative images were analyzed to quantify the fluorescence intensity values and calculate an average. Experiments were repeated 3 times individually at least.
For fluorescence intensity quantification, ImageJ was applied, and relative intensity was quantified by intensity divided by view area. For colocalization, the rate was quantified using ImageJ with colocalization finder. For each sample, no less than 5 representative images were analyzed to quantify the fluorescence intensity values and calculate an average. Experiments were repeated 3 times individually at least.