Zb 2305

Cell extracts containing 50 μg of protein were subjected to SDS-PAGE (Boster, Wuhan, China, AR0138) and transferred to PVDF membranes (Millipore, ISEQ00010). After blocking, membranes were incubated overnight with primary antibodies against SCP2 (1:1000), PKA (1:2500), SUFU (1:1000), GLI1 (Santa Cruz, sc-515751, 1:100), BCL2 (1:200), CCND1 (1:1000), p-AKT (1:1000), AKT (1:1000), p-mTOR (1:1000), mTOR (1:1000) and β-actin (1:5000). Membranes were probed with secondary antibodies (1:2000; Zsbio, Beijing, China, ZB-2305 and ZB-5301). The signals from the membrane were detected by enhanced chemiluminescence.

Western blotting was performed as previously reported [25 (link)]. Equal amounts of protein (up to 30 μg) from the treated mice were separated by 10% SDS–PAGE and transferred to PVDF membranes. Transferred blots were blocked with nonfat milk for 2 hours and then incubated overnight at 4 °C with primary antibody (1:1000). Blots were subsequently washed and incubated with goat anti-rabbit (ZB-2301, Zsgb-Bio, 1:5000) and goat anti-mouse (ZB-2305, Zsgb-Bio, 1:5000) secondary antibodies for 2 hours. The antibodies were listed in Table S1. Protein bands were detected with ECL reagent (WBKLS0500, Merck Millipore). Chemiluminescent signals were detected and analyzed using a Tanon-5500 chemiluminescent imaging system (Tanon Science and Technology Co., Ltd., Shanghai, P R China). The intensity of the bands was analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD, USA). Full-length blots/gels are presented in Supplementary Fig. S7.

The QPRT knockdown (QPRT siRNA), negative control (QPRT siRNA NC), and wildtype (control) HEK293T cells were seeded in 6-well plates. Cells were lysed in lysis buffer for 30 min on ice, and then insoluble materials were removed after 15 min centrifugation at 10,000 rpm/min. Protein concentrations were determined by BCA assay (Pierce, Rockford, United States). Lysate supernatant was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (IPVH00010, Millipore). The membrane was incubated with Rabbit monoclonal antibody against QPRT (ab171944; Abcam) or Mouse Anti-GAPDH antibody (1/2000; TA-08; ZSGB-BIO) followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (H + L) (1/2000; ZB-2301; ZSGB-BIO) and peroxidase-conjugated goat anti-mouse IgG (H + L) (1/2000; ZB-2305; ZSGB-BIO) secondary antibody, respectively. Western blots were developed using ECL solution. Grayscale results were analyzed by “Quantity one” software.

Western blot was performed as described previously. 24 (link) Specific proteins were detected using the following primary antibodies: NLRP3 (no. ab214185, 1:1000, Abcam), ASC (no. A1170, 1:1000, ABclonal), caspase-1 (no. ab238972, 1:1000, Abcam), IL-1β (no. ab9722, 1:1000, Abcam) and GSDMD (no. ab219800, 1:1000, Abcam). Horseradish peroxidase-conjugated secondary antibodies (ZB-2301, ZB-2305, 1:1000, ZSGB) were incubated with the membrane and the antibody complexes were detected by Imaging System (Bio-Rad, Hercules, CA, USA). β-Actin (no.TA-09, 1:1000, ZSGB) was used as the control to detect equal protein loading.

The cells were lysed with lysis buffer (Beyotime, Shanghai China) supplemented with a protease inhibitor solution (Beyotime) and centrifuged at 12 000 g for 10 min at 4 °C. Extracted proteins were separated on 10–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 37 °C, the membranes were incubated overnight with primary antibodies at 4 °C, washed with TBST, and then incubated with secondary antibodies. The western blot results were quantified by densitometric analysis using the Quantity One 4.6.9 software (Bio-Rad).
The following antibodies were used: anti-cleaved caspase-9 (9509T, CST), anti-cleaved caspase-3 (9664T, CST), anti-Bax (#2772, CST), anti-Bcl2 (#3498, CST), anti-cytochrome c (10093, Proteintech), VDAC rabbit mAb (4661, CST), anti-MFN1 (NBP1-71775, Novus Biologicals), DRP1 rabbit mAb (8570, CST), OPA1 rabbit mAb (80471, CST), anti-FIS1 (D122377-0025, BBI life sciences), anti-MFN2 (12186, Proteintech), anti-beta tubulin (10094, Proteintech), anti-PSD95 (20665, Proteintech), spinophilin rabbit mAb (14136, CST, Boston), HP-goat anti-mouse (ZB-2305, Zsbio, Beijing, China), HP-goat anti-rabbit (ZB-2301, Zsbio, Beijing, China), and Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (33112ES60, Yeasen).

A total of 4–5 single colonies were inoculated in 3 mL LB medium and incubated at 37°C overnight. Cell pellets from 2 mL of overnight cultures were collected by centrifugation at 8,000 rpm for 10 min, washed three times with physiological saline, and resuspended in 200 μL of radioimmunoprecipitation (RIPA) lysate (Beyotime, cat no. P0013C). The cell suspensions were sonicated for 1.5 min (work 3 s, stop 6 s, with frequency of 28 KHz), centrifuged at 14,000 rpm, 4°C for 10 min, and the supernatants were collected. Equal amounts of proteins (100 μg/lane) from each bacterial lysate were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare). Blots were probed with the Polymyxin resistance protein MCR-1 primary antibody (polyclonal mouse anti-E. coli polymyxin resistance protein MCR-1 antibody; LS Bio), followed by horseradish enzyme labeled goat antimouse IgG (ZB-2305; ZSGB-Bio). Protein bands were visualized using an enhanced chemiluminescence (ECL) detection method (Bio-Rad), and band intensities were analyzed with a densitometer (LAS-4000; GE Healthcare). The experiment was repeated 3 times. OmpA, measured quantitatively using an OmpA antibody (polyclonal rabbit anti-Salmonella typhi OmpA antibody; LS Bio), followed by horseradish enzyme labeled goat anti-rabbit IgG (ZB-2301; ZSGB-Bio), was used as the control.