Ab45134

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).

For sample preparation, adherent cells after photocatalytic labeling were directly scrapped from plate using RIPA strong lysis buffer (120 μL/well) (CWBIO, CW2333S) supplemented with 1× protease inhibitor cocktail (Bimake, B14001) and transferred to 1.5-mL tube, while suspension cells were pelleted by centrifugation (300–500 g, 3 min, 4 °C) and lysed in the same buffer. The samples were incubated on ice for 10 min, followed by the addition of methanol and chloroform (lysate:methanol:chloroform = 4:4:1, v/v/v) to precipitate proteins for purification. After cooling the mixture for >30 min at −80 °C, the proteins were pelleted by centrifugation (10,000 g, 20 min, 4 °C), carefully washed with cold methanol (200 μL) twice, and redissolved in 1% SDS-PBS buffer (80 μL). Five times SDS-PAGE loading buffer (20 μL) (Beyotime) was added to the sample, followed by heating at 95 °C for 15 min. Immunoblotting was performed with Tris-glycine SDS-PAGE and 0.2 μm PVDF membrane. Mouse anti-biotin mAb (Santa Cruz, sc-101339, 1:1,000 dilution) and rabbit anti-HSP60 mAb (Abcam, ab45134, 1:2,000 dilution) were used as primary antibodies, while HRP-linked anti-mouse IgG (Cell Signaling Technology, 7076S) and HRP-linked anti-rabbit IgG (Cell Signaling Technology, 7074S) were used as secondary antibodies (1:5,000 dilution).

The following commercial antibodies were used in this study: anti-β-actin (mouse; Sigma, A1978; RRID:AB_476692), anti-emerin (rabbit, abcam Ab40688, RRID:AB_2100059), anti-HA (mouse, Covance, MMS-101P, RRID:AB_2314672), anti-lamin A/C (mouse, ImmuQuest (IQ332 RRID:AB_10660272)), anti-lamin B1 (rabbit, abcam ab16048, RRID:AB_443298), anti-lamin B2 (rabbit, abcam (ab151735) RRID:AB_2827514), anti-LAP1 (rabbit, abcam, ab86307 RRID:AB_2206124), anti-LBR (rabbit; Abnova, PAB15583; RRID:AB_10696691), mAB414 (mouse; Abcam, ab 24609; RRID:AB_448181), anti-H3 (rabbit, acam ab1791, RRID:AB_302613), anti-Tor1A (rabbit, Abexxa, abx001683), anti-Tor1B (rabbit, antibodies-online, ABIN1860834), anti-HSP60 (rabbit, abcam, ab45134, RRID:AB_733033), anti-α-tubulin (mouse, Sigma, T5168, RRID:AB_477579).
Antibodies were raised in rabbits against purified, recombinant human LULL1. Antibodies against human SUN1 (Sosa et al., 2012 (link)), human SUN2 (Turgay et al., 2010 (link)) and anti-GFP (Turgay et al., 2014 (link)) have been previously described.