Fitc conjugated goat anti mouse igg

Cells coated onto glass coverslips were gently washed 3 times in warm PBS, fixed in tissue cell fixative solution (Biosharp, Beijing, China) for 10 min at room temperature (RT), permeabilized in 0.1% Triton X-100 (diluted in PBS; Biosharp) at RT for 20 min and blocked in 5% bovine serum albumin (in PBS) at RT for 2 h. The cells were then separately incubated with primary antibody against ASC (1:100 dilution) or NLRP3 (1:100 dilution) at 4 °C overnight, and secondary antibody of Cy3-labeled goat anti-rabbit IgG(H+L) (1:400; EarthOx, San Francisco, CA, USA) or FITC-conjugated goat anti-mouse IgG (1:400; Earthox) at 37 °C overnight for 1 h in the dark. Cell nuclei were stained with Hoechst 33258 (10 µg/ml; UE, Suzhou, China) for 5 min and observed under a fluorescence microscope (Olympus Corp., Tokyo, Japan).

A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). The antibodies against phospho-Akt (Ser473), Akt, phospho-JNK (Thr183/Tyr185), JNK, phospho-p38, p38, phospho-NF-κB (Ser536), NF-κB, phospho-IκBα and IκBα were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-ERK1/2, ERK1/2, caspase-3, caspase-8, caspase-9, CytoC and GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA). Beta-Tubulin, HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Beyotime (Jiangsu, China). LY294002 (PI3K/Akt inhibitor), PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) and Bay11-7082 (NF-κB inhibitor) were purchased from Selleck (Houston, USA). FITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG were purchased from EarthOx Life Sciences (Millbrae, CA, USA). The 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Jiangsu, China).