Rabbit anti mouse asc ab 67 824

Cells were cultured in a 12-well plate and infected with P. multocida as described above. After incubation, the supernatants of PEC and neutrophils were collected and cells were lysed with radio‐immunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China). These samples were concentrated with 20% (w/v) trichloroacetic acid (TCA) and the concentration was determined using a BCA protein detection kit (Beyotime). The supernatants and cell lysates were separated by a 10–15% SDS-PAGE gel and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with indicated antibodies (Abs) containing mouse anti-mouse caspase1-p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse gasdermin D (GSDMD) Ab (ab209845) (Abcam, Cambridge, UK), rabbit anti-mouse ASC Ab (67,824) (Cell signaling technology, Danvers, MA, USA), mouse anti-rabbit GAPDH Ab (AG019-1) (Beyotime), rabbit monoclonal anti-mouse histone H3 (citrulline R17) (ab219407) (Abcam, Cambridge, UK). Finally, the distinct protein bands were detected by ECL detection reagent (Beyotime). The bands in western blot were quantified into numerical values using image J software.

Cells were prepared in 12-well plates and infected with PmCQ2 as described above. After the indicated time of infection, the supernatants were collected and concentrated using 20% (w/v) trichloroacetate (TCA), and then cells were lysed with 1 × SDS loading buffer (Beyotime, Beijing, China). Next, the supernatants and cell lysates were separated by 10–15% SDS‒PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA). Finally, distinct protein bands were detected by ECL detection reagent (Biosharp, China). The Western blotting bands were quantified to obtain numerical values using image software.