Anti cleaved caspase 1 asp296

Protein extracts (50 µg) were fractionated by 10% SDS-PAGE and then transferred to PVDF membranes. The blots were washed with wash buffer (PBS1x, 0.05% Tween20), blocked for 1 h with 0.1% BSA, and then incubated with primary antibodies anti-cleaved Caspase-1 (Asp296) from Cell Signaling and anti-cleaved Caspase 3 (Asp175) (cat nº 96645) from Cell Signaling. The protein levels were expressed as the ratio between the target protein and ß-actin or tubulin detected in the same membrane.

The following antibodies were used: anti-NLRP3 (catalog number 15101), anti-Akt (catalog number 4691), anti-phospho-Akt (Ser473) (catalog number 4060), anti-LC3B (catalog number 2775), anti-IL-1β (catalog number 12242), anti-GSDMD (catalog number 39754), anti-cleaved-caspase-1 (Asp296) (catalog number 89332), anti-cleaved-caspase-1 (Asp297) (catalog number 4199), anti-phospho-mTOR (ser2448) (catalog number 5336), and anti-mTOR (catalog number 2972) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-p62 (catalog number A11250), Alexa Fluor 488 goat anti-rabbit IgG (AS053), and anti-AIM2 (catalog number A3356) were obtained from ABclonal Technology (Wuhan, China); antibodies against ASC (catalog number sc-514414) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); and anti-β-actin (catalog number 66009-1-lg) monoclonal antibody and caspase-1 (catalog number 22915-1-AP), and anti-CHMP2A (catalog number 10477-1-AP) were acquired from Protein Tech Group (Chicago, IL).

Rat kidney tissues were fixed in formalin, then paraffin embedded, and 5 μm sections were mounted on silanized slides. Immunodetections were performed while using primary anti-αSMA (sc130617) from Santa Cruz Biotechnology, anti-cleaved Caspase-1 (Asp296) (cat nº 673145), and anti-cleaved Caspase 3 (Asp175) (cat nº 96645) from Cell Signaling and anti-NFκB-p65 (ab16502) from Abcam in blocking solution overnight at 4 °C. Immunosignals were visualized while using the LSAB+ System–HRP (DakoCytomation, Carpinteria, CA, USA). Counterstaining were done using Hematoxylin and eosin. The cellular distribution of Caspase-1 in the kidney was established when comparing with the pattern of staining of aquaporin-1 and aquaporin-2, as previously discussed [31 (link)]. For immunofluorescent staining, the sections were incubated with anti-NFκB-p65 (ab16502) from Abcam and Alexa Fluor 488-conjugated secondary antibodies (Zhongshan Golden Bridge Biotech, Beijing, China) Nuclei were stained while using DAPI. Images were captured using a fluorescence microscope Leyca DM1000LED. Images were analyzed using the software ImageJ with Color Deconvolution plugin (National Institute of Health, USA).