Anti β tubulin

At the end of capsaicin and SMF treatments, cells were washed with PBS and fixed with 3.7% paraformaldehyde (PFA; Sigma) in PBS for 15 min at RT. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 15 min. Nonspecific protein binding was blocked with 1% BSA in PBS for 20 min at RT. After washing three times with PBS, the cells were incubated with anti-β-tubulin (1:1000 dilution; Abcam) and anti-active caspase-3 (1:800 dilution; Cell Signaling Technology) primary antibodies overnight at 4°C. The cells were then washed three times in PBS, and incubated in the Alexa Fluor 488-conjugated donkey anti-goat secondary antibody and the Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (1:500 dilution; Jackson ImmunoResearch Inc.) for 1 h at 37°C in the dark. The coverslips were mounted to slides in mounting medium with DAPI (Abcam). The mounted samples were examined by a Zeiss LSM 880 inverted laser scanning confocal microscope. Pictures of randomly selected areas were taken for each sample and representative micrographs are shown in the figures.

Western blot analysis was carried out as previously described [29 (link)]. Lysates were fractionated by SDS-PAGE and proteins were blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk and then incubated overnight at 4 °C with antibodies to anti-FASN (Sigma-Aldrich, St. Louis, MO, USA), anti-DHCR24 (Cell Signaling Technology, Danvers, MA, USA), p27 kip1 (BD Biosciences, Franklin Lakes, NJ, USA) and Bim (Abcam, Cambridge, UK). Anti-actin (Sigma-Aldrich, St. Louis, MO, USA) or anti-β-tubulin (Abcam, Cambridge, UK) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (GE Healthcare, Chicago, IL, USA). Secondary antibodies were obtained from GE Healthcare. At least three independent experiments were performed. Band intensity quantification was performed using the ImageJ 1.47 v software.

Cells were seeded in six-well plates at a concentration of 5 × 10⁵ per well in 1 mL of medium and transfected with miRNA mimics or miRNA inhibitors. The cells were stimulated with type I IFN (1000 U/mL) 24 h after transfection and the cells were lysed at the indicated time points. The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively). The secondary antibodies used were obtained from Cell Signaling: horseradish-peroxidase (HRP)-linked anti-rabbit IgG antibody (diluted 1:5000), and HRP-linked anti-mouse IgG antibody (diluted 1:4000). The signal was generated with SuperSignal® West Pico Luminol/Enhancer Solution (Pierce). We used Tanon 6600 Luminescent Imaging Workstation (Tanon Science & Technology, Shanghai, China) to scan the film. And band signal intensity was analyzed using Image J.