Ab108362

Single retinas were homogenized and sonicated in lysis buffer and centrifuged. Sample buffer was added to the supernatant just prior to use. Known amounts of protein (10 to 20 μg) or protein ladder were loaded into each well of an SDS-polyacrylamide gel. The Bio-Rad mini-trans blot cell system and mini protean pre-cast gells at 4–20% were used (Hercules, CA). Loading controls included GAPDH (1:1000; ab9485, Abcam, Cambridge, MA) or HSP60 (1:1000; ab45134, Abcam). The protein was transferred onto nitrocellulose using the Bio-Rad trans blot turbo transfer system (Hercules, CA) and alkaline phosphatase was used for band detection. Band density was quantified by scanning the blot using an EPSON scanner and Adobe Photoshop to convert to grayscale and invert the image. Each band was selected with the same frame and set measurements were used to obtain the greay mean value for each. Antibodies used were anti-caspase 1 (1:1000; ab108362, Abcam), anti-SOD2 (1:1000; ab13533; Abcam), anti-TXNIP (1:1000; 14715, Cell Signaling Technology, Danvers, MA), anti-NLRP1 (1:1000; 4990, Cell Signaling Technology), and anti-NLRP3 (1:1000; 15101, Cell Signaling Technology).

Hearts were homogenized in RIPA lysis buffer (9806; Cell Signaling). Protein concentration was determined by BCA kit assay (23225; Pierce Biotechnology). An equal amount of protein (30 μg) was separated by sodiumdodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio‐Rad). Subsequently, the membrane was blocked with 5% nonfat dry milk or 3% bovine serum albumin (37520; Thermo Fisher Scientific) in TBST for 1 h (room temperature). Primary antibodies for NLRP3 (ab4207; Abcam, 1:500), ASC (sc22514; Santacruz, 1:500), caspase‐1 (ab108362; Abcam, 1:500), IL‐1β (ab9722; Abcam, 1:500), eNOS (#32027; Cell Signaling, 1:500), NOX2 (ab129068; Cell Signaling, 1:1000), NOX4 (ab133303; Abcam, 1:5000), and β‐actin (sc47778; Santa Cruz Biotechnology, 1:2000) were incubated at 4° overnight, and those were matched to their corresponding horseradish peroxidase‐conjugated secondary antibodies for 1 h (room temperature). Blots were developed with the enhanced chemiluminescence (RPN2232; GE Healthcare Life Science), scanned with the densitometer (FluorChem IS8900; Alpha Innotech, Santa Clara, CA), and quantified using NIH‐ImageJ software. Data were normalized with the corresponding internal reference β‐actin. Relative value was normalized to wild‐type control, LF‐SED.

Protein extractives were lysed in mammalian cell lysis buffer. Protein concentration was determined referring to a bovine serum albumin standard. Proteins were separated on 10–12% SDS-PAGE gels. Protein expression levels were determined using primary antibodies anti-NLRP3 (1:500, ab214185), anti-caspase-1 (1:500, ab108362), anti-IL-1β (1:500, ab2105), anti-HAS1 (1:500, ab198846), anti-p-p38 (1:500, ab45381) and anti-p38 (1:500, ab31828), which were purchased from Abcam (Cambridge, UK), and primary antibodies anti-CD44 (1:1000, 15675–1-AP), anti-OPN (1:1000, 22952–1-AP) were purchased from Proteintech (USA). Anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz (USA). GAPDH (Santa Cruz, USA) was used as loading control.