Vimentin monoclonal antibody

Vimentin monoclonal antibody was purchased from Abcam (Cambridge, UK). Influenza virus NP protein monoclonal antibody was made by our laboratory [19 (link)]. GAPDH monoclonal antibody was purchased from Enogene Biologicals (Nanjing, China). β-actin monoclonal antibody, AMPK monoclonal antibody and AMPK phosphorylated monoclonal antibody P-AMPK were purchased from BOSTER Biologicals (Nanjing, China). HMGCR monoclonal antibody and HMGCR phosphorylated monoclonal antibody P-HMGCR were purchased from Abmart Biologicals (Shanghai, China). HRP-labeled goat anti-rabbit/mouse IgG secondary antibody was purchased from Univ Biologicals (Shanghai, China). Lipid raft disruptor MβCD, water-soluble cholesterol and cholesterol inhibitor lovastatin were purchased from Sigma Aldrich (Burlington, MA, USA). Total cholesterol (TC) content assay kit and puromycin were purchased from Sangon Biotech (Shanghai, China).

To analyze protein expression by regular or confocal fluorescence microscopy, we used standard protocols previously described [23 (link),25 (link)]. Cells were seeded on 2% gelatin-coated glass coverslips in DMEM supplemented with 10% FBS. For vimentin analysis, cells were fixed with 4% paraformaldehyde in PBS for 20 min at RT and incubated with a blocking solution containing 5% BSA and 0.3% Triton X-100 in PBS for 1 h at RT. Primary antibody incubation (Vimentin monoclonal antibody from Abcam (Cambridge, UK), Ab45939; 1:200 in 0.1% BSA-PBS) was performed for 15 h at 4 °C. Anti-mouse Alexa Fluor 488 conjugated secondary antibody (A11012, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) was diluted 1:200 in PBS 1% BSA and applied for 1 h at RT. Antibody incubations were carried out in a humidity chamber to avoid evaporation. For visualization, cells were embedded in Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting medium with DAPI (Sigma-Merck) and visualized using a Leica SP-8 fluorescence microscope.