Rabbit anti gsdmd

As previously described by us,
⁴⁰ (link) proteins were extracted from the MCA‐supplied brain regions and loaded onto SDS‐polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti‐NLRP3, Merck; 1:500, rabbit anti‐ASC, Abcam; 1:1000, rabbit anti‐IL‐1β, Abcam; 1:1000, rabbit anti‐IL‐18, Proteintech; 1:1000, rabbit anti‐caspase‐1, Abcam; 1:500, rabbit anti‐GSDMD, Affinity; 1:2000, rabbit anti‐G3BP1, Proteintech; 1:500, rabbit anti‐TIA1, Proteintech; 1:1000, rabbit anti‐DDX3X, Proteintech; 1:1000, rabbit anti‐IgG antibody, Cell Signaling Technology; 1:10,000, rabbit anti‐SYN, Abcam; 1:1000, rabbit anti‐PSD‐95, Cell Signaling Technology; 1:1000, rabbit anti‐BDNF, Abcam; 1:500, rabbit anti‐Ang‐1, Proteintech; 1:500, rabbit anti‐Ang‐2, Proteintech; 1:500, rabbit anti‐VEGF, Abcam; 1:1000, mouse β‐actin, Santa Cruz) for 24 h at 4°C. Next, membranes were incubated with a secondary antibody (1:4000, goat anti‐rabbit IgG, goat anti‐mouse IgG, Cell Signaling Technology) for 2 h at room temperature. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health) to quantify protein expression in terms of relative image density.

The right brain cortex and retina protein samples (10 μg) were subjected to 8–12% SDS-PAGE and then transferred to PVDF membranes (Merk Millipore, Burlington, MA, USA). Blocked with 5% normal goat serum (NGS) for 2 h, the membranes were incubated with primary antibodies: rabbit Anti -NLRP3 (1:1000, Cat# ab263899; Abcam), rabbit anti-GSDMD (1:500, Cat# AF-4012; Affinity), rabbit anti-caspase-1 (1:1000, Cat# AF-4022; Affinity), and rabbit anti-β-actin (1:200; Cat# AF5003; Beyotime Biotechnology, Shanghai, China) at 4 °C for 24 h. The membranes were then incubated with anti-rabbit secondary antibodies (1:500; Cat# A0239; Beyotime Biotechnology, Shanghai, China) at 4 °C for 2 h. The protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Merk Millipore, Burlington, MA, USA) and analyzed using ImageJ analysis software (https://imagej.net/Fiji/Downloads, accessed on 30 May 2017, Bethesda, MD, USA).