Hrp 60008

Proteins in kidney tissues and HK2 cells were extracted using RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China) containing 1% PMSF (P0100, Solarbio, Beijing, China). The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (R7CA6580A, Thermo Fisher Scientific, Waltham, MA). The membranes were probed overnight with GPX4 (1:1000, ab125066, Abcam, Cambridge, MA), and NCOA4 (1:1000, abs-134557, Absin, Shanghai, China). The membranes were respectively incubated with HRP-labeled goat anti-rabbit IgG (1:5000, 511203, Zen BioScience, Research Triangle Park, NC) and HRP labeled goat anti-mouse IgG (1:5000, 511103, Zen BioScience). Bands were detected using Immobilon Western Chemilum HRP Substrate (Catalogue number WBKLS0500, EMD Millipore, Burlington, MA), and protein levels were normalized against β-actin (1:5000; HRP-60008, Proteintech, Rosemont, IL).

Male germ cells from wild-type mice and P63^(+/−) mice were lysed with RIPA buffer (BiotechWell, Shanghai, China) for 30 min on ice. Cell lysates were cleared by centrifugation at 12,000×g for 15 min at 4 °C, and the protein concentrations were measured by BCA kit (DingGuo ChangShengBiotech, Beijing, China). In total, 30 mg of cell lysate from each sample were separated using 10% SDS-PAGE (Bio-Rad Laboratories) and transferred to nitrocellulose membranes for 2 h at room temperature. The membranes were blocked using 5% nonfat dry milk in TBS-T for 1 h at room temperature. After washing with TBS-T, the membranes were incubated with antibodies against P63 (Abcam, ab735, 1:500) and ACTB (Protein tech, catalog no: HRP-60008, dilution: 1:5000) overnight at 4 °C. After extensive washes, the membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (IgG) (Santa Cruz Biotechnology) at a 1:2000 dilution for 1 h at room temperature. The membranes were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA, USA), and densitometric analyzes were processed with Adobe Photoshop 8.0. The relative level of P63 protein was normalized to the expression of ACTB.

Lysates were prepared from cardiac tissue or NRVMs by fine homogenization or cell scraping in radioimmunoprecipitation assay buffer with a phosphatase–protease inhibitor cocktail (Roche) on ice. Lysates were centrifuged and protein concentration was assessed by BCA assay. Samples were resolved on a 10% reducing sodium dodecyl sulfate (SDS) polyacrylamide gel and blotted on a PVDF membrane. Membranes were blocked for 1 h at room temperature in 3% bovine serum albumin for phospho-antibodies or 5% low-fat milk for other antibodies in Tris-buffered saline and 0.1% Tween-20 (TBS-T). Primary antibodies were incubated overnight at 4°C. Antibodies used were: total-Pyk2 (CST 3292S, 1:1000); phospho-Pyk2 Y402 (Abcam 4800, 1:1000); phospho-Pyk2 Y579/580 (Invitrogen 44-636G, 1:1000); AE2 (Novus NBP159858, 1:500); and NHE1 (BD Biosciences 61175, 1:500). Membranes were washed with TBS-T and incubated with anti-rabbit/mouse HRP-conjugated secondary antibody (GE Healthcare Lifesciences). For loading controls, actin HRP-conjugated (Proteintech HRP60008, 1:20 000) and GAPDH HRP-conjugated (Proteintech HRP60004, 1:10 000) were used. Antibody–antigen complexes were visualized by Pierce™ enhanced chemiluminescent substrate with a Bio-Rad ChemiDoc™ Imaging System.

The total protein content was extracted from hippocampal tissue or cultured cells using RIPA lysis buffer (Beyotime, China) containing PMSF (BioSharp, China) and phosphatase inhibitor (Beyotime, China). The protein concentration in the supernatant was quantified by a BCA protein assay (Beyotime, China). Equal amounts of protein samples were then separated by SDS–PAGE and transferred to PVDF (MilliPole, UK). PVDF membranes were then blocked with 5% BSA for 2 h at room temperature and incubated with primary antibodies (Erbin (NBP2-56,104, Novus), NLRP3 (A5652, Abclonal), GSDMD (A10164, Abclonal), Caspase-1 (AG-20B-0042, adipogenic), Iba-1 (ab178846, Abcam), IRE1α (3294, CST), p-IRE1α (ab48187, Abcam), Xbp1s (24,868–1-AP, proteintech), PSD95 (3409, CST), Synaptophysin (AF0257, affinity), Synapsin-1 (5297, CST), β-actin (HRP-60008, proteintech)) antibody overnight at 4 °C. After incubation, membranes were washed with TBST and then incubated with the corresponding HRP-conjugated secondary antibodies for 2 h. Then, washed the membrane with TBST. Finally, immunoreactive bands were detected with an enhanced chemiluminescence detection reagent. Band intensities were quantified by spot densitometric analysis using ImageJ software, and results were normalized to β-actin levels and reported as relative intensities to controls.

Cell lysates were acquired by treating cells with RIPA lysis buffer (20-188, Millipore), and the protein concentration was determined using a Bio-Rad Protein Assay Kit (5000002, Bio-Rad). These lysates were then loaded onto a 12% polyacrylamide gel and transferred onto a PVDF membrane (GE Healthcare). For immunolabelling, primary and secondary antibodies were diluted in PBST with 5% milk and left to incubate overnight. The antibody labelling was visualised using the SuperSignal West Pico PLUS Chemiluminescent Substrate (34579, Thermo Scientific) and captured with a chemiluminescence imaging system (GeneGnome XRQ, Syngene). The antibodies used included rabbit anti-IMPDH2 polyclonal antibody (1:10000, 12948-1-AP, ProteinTech), rabbit anti-phospho-Akt1 (S473) monoclonal antibody (1:3000, AP0637, Abclonal), rabbit anti-Akt (Pan) polyclonal antibody (1:3000, IR171-666, iREAL Biotechnology), mouse anti-OCT3/4 monoclonal antibody (1:3000, sc-5279, Santa Cruz Biotechnology), HRP-conjugated mouse anti-β-ACTIN monoclonal antibody (1:3000, HRP-60008, ProteinTech), mouse anti-α-TUBULIN monoclonal antibody (1:10000, T5168, Sigma-Aldrich), HRP-conjugated goat anti-mouse IgG polyclonal antibodies (1:10000, 31430, Invitrogen) and HRP-conjugated goat anti-rabbit IgG polyclonal antibodies (1:10000, 31460, Invitrogen).

For preparation of cell lysate, cells were lysed in RIPA buffer (1% (v/v) Igepal CA-630, 0.5% (w/v) sodium deoxycholate and 0.1% (w/v) sodium dodecyl sulphate (SDS) in phosphate-buffered solution (PBS) containing aprotinin and sodium orthovanadate. Aliquots of cell lysate containing 10 µg of protein were resolved by 10% (w/v) SDS–polyacrylamide gel electrophoresis, followed by electrotransfer to a PVDF hybond (Amersham, UK) membrane. Immunodetection was carried out using antibodies anti-PARP (#9542 Cell Signaling; 1:1000), anti-cleaved Caspase-3 (#9661 Cell Signaling; 1:1000), anti-TRAIL (#3219 Cell Signaling; 1:2000), anti-DR4/TRAIL-R1 (#42533 Cell Signaling; 1:1000), anti-DR5/TRAIL-R2 (#8074 Cell Signaling; 1:2000), anti-Actin-HRP (HRP-60008 Proteintech; 1:30,000), anti-GAPDH-HRP (HRP-60004 Proteintech; 1:30,000) and anti-rabbit-HRP (111-035-144 Jackson Immuno Research; 1:10,000), which were detected using a chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK).