β tublin 9f3

Lung tissues were homogenized in ice-cold lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Sigma-Aldrich, USA) for 40 min, then centrifuged at 12,000 g for 20 min. Protein concentrations were measured using the BCA assay. 30 μg of total protein was sampled and separated in 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were then blocked with 5% fat-free milk in Tris-buffered saline and 0.5% Tween-20 (TBS-T) for 2 h and then incubated with corresponding primary antibodies overnight at 4 °C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (ZB2306) for 1 h at room temperature. Finally, signals were gained by using the enhanced chemiluminescence (ECL) kit (CW Biotechnology, Beijing, China) and were exposed by using a biological molecular imaging instrument (LAS4000mini, JAPAN). The immunoblots intensities were quantified using Quantity One software (Bio-Rad, CA, USA). The primary antibodies including P38 (D13E1), p-P38 (D3F9), ERK (137F5), P-ERK (D13.14.4E), JNK (56G8), p-JNK (98F2), β-Tublin (9F3) and GAPDH (14C10) were purchased from Cell Signaling Technology, Inc. (CST, MA, USA).

We used rabbit polyclonal GFP (Invitrogen), mouse monoclonal MAP2 (Sigma-Aldrich), mouse monoclonal SMI-312R (Covance), mouse monoclonal Myc (Cell Signaling, 9B11), rabbit monoclonal β-tublin (9F3, Cell Signaling), Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen), and HRP-conjugated secondary antibodies (GE Healthcare), as well as CL075 (InvivoGen) and poly dAdT (InvivoGen). poly dAdT is known to activate AIM2 inflammasomes14 (link)55 (link)56 (link). The length of poly dAdT used in this report is 1184 bps.