Horseradish peroxidase conjugated goat anti rabbit secondary antibody

Cells were lysed in lysis buffer (Beyotime) containing complete protease mixture (Sigma-Aldrich). After centrifugation, the lysates were boiled in SDS loading buffer and resolved by 12% acrylamide gel electrophoresis in the presence of SDS (SDS-PAGE), then transferred to a PVDF membrane (Millipore) and probed with 1:1000 dilution of a rabbit anti-p38 or anti-beta-actin polyclonal antibody (Santa Cruz Biotechnology), followed by 1:1000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Promega). Then the polypeptides were revealed using ECL reagent (Amersham Biosciences). All of the figures illustrating Western blotting analyses are representative of at least three independent experiments.

Cell lysates were extracted from BV-2 cells using RIPA lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Roche, Germany). The lysates were then run on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Primary antibodies used for Western blotting included monoclonal rabbit anti-cGAS (#31659S, Cell Signaling Technology, USA), monoclonal rabbit anti-phospho-tank binding kinase 1 (TBK1)/NAK (Ser172) (#5483S, Cell Signaling Technology, USA) and monoclonal rabbit anti-TBK1/NAK (#ab40676, Abcam, USA). Following incubation with the primary antibodies, horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Promega, USA) and the WesternBright ECL reagent (Advansta, USA) were sequentially applied. The blots were scanned using a ChemiScope 6000 imaging system (Clinx Science Instruments, China). Densitometry analysis was conducted using ImageJ.