Sqstm1

Immunoprecipitation and Western blot assays were performed according to the previously described procedures [4 (link)]. Antibodies Runx2 (Abcam, UK, 1:1000), ALP (Abcam, UK, 1:2000), SM22ɑ (Abcam, UK, 1:1000), p16 (Proteintech, USA, 1:500), p21 (Cell signaling, USA, 1:1000), LC3-II (Abcam, UK, 1:500), SQSTM1 (Abcam, UK, 1:1000), Atg10 (Abcam, UK, 1:1000), Bhlhe40 (Proteintech, USA, 1:500), GAPDH (Abcam, UK, 1:4000), and β-actin (Proteintech, USA, 1:2000) were used in this study. HA-VSMCs were transfected as indicated and lysed in an immunoprecipitation buffer. The lysates were centrifuged for 20 min. Supernatants were incubated with specific antibodies overnight at 4℃ and protein A/G Agarose beads for 4 h. The immunoprecipitates were washed three times with PBS, and then analyzed via SDS-PAGE. The protein bands were visualized using ECL-Plus Western blot detection kit (Amersham BioSciences, UK).

Immunoblotting procedures were performed as we described previously. Briefly, the heart samples were homogenized in the lysis buffer. Lysates were clarified at 12,000 g for 10 min at 4°C, and protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were loaded onto 8–12% SDS–PAGE, transferred onto polyvinylidene difluoride membranes, probed with the indicated primary antibody and the appropriate secondary antibody conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA), and the immune complexes were detected by standard methods. The antibodies used in this study were as follows: GAPDH (Cell Signaling, Danvers, MA), p-P65 (Cell Signaling, Danvers, MA), P65 (Cell Signaling, Danvers, MA), p-ULK1 (Cell Signaling, Danvers, MA), ULK1 (Cell Signaling, Danvers, MA), p-Beclin1 (Cell Signaling, Danvers, MA), Beclin1 (Cell Signaling, Danvers, MA), LC3 (Cell Signaling, Danvers, MA), p-MTOR (Cell Signaling, Danvers, MA), MTOR (Cell Signaling, Danvers, MA), NLRP3 (Abcam, Cambridge, MA), SQSTM1 (Abcam, Cambridge, MA). HRP linked anti-rabbit or anti–mouse IgG second antibodies (Cell Signaling, Danvers, MA).

P-PDK1(Ser241), Akt, p-Akt(Ser473), 4EBP1 and p-4EBP1(Ser65) and α-tubulin were purchased fromCell Signaling Technology (Danvers, MA, USA). P70S6K and p-P70S6K(Thr470) were acquired from Millipore (Boston, MA, USA). Antibodies against LC3, SQSTM1, Atg5, Beclin1, LAMP1, Bcl-2, Bax, cytochrome C, active caspase-9, active caspase-3, cleaved PARP, mTOR, p-mTOR(S2448) and β-actin were obtained from Abcam (Cambridge, UK). YZT was dissolved in DMSO and diluted with culture medium. CQ, 3-methyladenine (3-MA) and rapamycin (RAPA) were bought from Sigma-Aldrich (Saint Louis, MO, USA).

The following antibodies were used: CCR5 (Cat# ab65850, RRID:AB_1140936), CXCR4 (Cat# ab124824, RRID:AB_10975635), and SQSTM1 (Cat# ab56416, RRID:AB_945626) from Abcam, CD4 (Cat# 300501, RRID:AB_314069) from BioLegend, ATG5 (Cat# 2630, RRID:AB_2062340), ATG7 (Cat# 2631, RRID:AB_2227783), BIRC2 (Cat# 7065, RRID:AB_10890862), CASP8 (Cat# 9746, RRID:AB_2275120), PARP1 (Cat# 9532, RRID:AB_659884), RIPK1 (Cat# 3493, RRID:AB_2305314), XIAP (Cat# 2045, RRID:AB_2214866) from Cell Signaling Technologies, MAP1LC3B (Cat# NB100-2220) from Novus Biologicals, and ACTB (Cat# A2228, RRID:AB_476697) from Sigma. Whole cell lysates and TNP membranes were prepared, resolved, and proteins detected as previously described 10 (link), 52 (link). Relative densities of the target bands were compared to the reference (ACTB) and were calculated using Fiji from the Max Planck Institute of Molecular Cell Biology and Genetics (Fiji, RRID:SCR_002285) 53 (link).

Antibodies recognizing the following proteins were used in this study: caspase-3, Bax, LC3, ATG7, COXIV, β-actin, and HRP-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA); pro-IL-1β, IL-1β, PGC-1α, TFAM, and SQSTM1 (Abcam, Cambridge, MA); MyHC (R&D Systems, Minneapolis, MN); Nlrp3 and ASC (AdipoGen, San Diego, CA); pro-IL-18 and IL-18 (MBL, Minneapolis, MN); and procaspase-1 and caspase-1 (Santa Cruz Biotechnology, Santa Cruz, CA). The SOD2 assay kit, MDA assay kit, and ATP assay kit (Beyotime Biotechnology, China) were also used.

The liver tissues or cell lysates of the operation group and the control group were extracted with cell lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 20 mM Tris (pH 7.4), 137 mM Nacl). The protein was subjected to 10% or 12% SDS-PAGE electrophoresis and then transferred to PVDF nitrocellulose membrane. Antibodies against ATP6V0D2 (Sigma-Aldrich), Hes1 (Cell Signaling Technology), NLRP3, IL-1β, ASC, LC3B, SQSTM1, and β-actin (Abcam) were used for detection.