P enos

Rabbit polyclonal anti-LRRC8A antibody was generated against the epitope QRTKSRIEQGIVDRSE (Pacific Antibodies) (Kang et al., 2018 (link)). All other primary antibodies were purchased from Cells Signaling: anti-ß-actin (#8457), Total Akt (#4685S), Akt1 (#2938), Akt2 (#3063), p-eNOS (#9571), Total eNOS (#32027), p-AS160 (#4288), p-p70 S6 Kinase (#9205S), pS6 Ribosomal (#5364S), GAPDH (#5174), pErk1/2 (#9101), Total Erk1/2 (#9102). Anti-LRRC8A antibody was custom made as described previously (Zhang et al., 2017 (link); Kang et al., 2018 (link)). A second p-eNOS antibody was purchased from Invitrogen (Cat#PA5-104858) and used to compare with anti-p-eNOS from Cell Signaling (#9571) for p-eNOS IF staining. Purified mouse anti-Grb2 was purchased from BD (610111) and Santa Cruz (#sc-255). Rabbit IgG was purchased from Santa Cruz (sc-2027). Anti-CD31 was purchased from Thermo Fisher (MA3105).

CRIF1, GCH1, SPR, DHFR, and p16 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTS antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). P-eNOS, T-eNOS, P-Akt, and T-Akt antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). Whole HUVECs and lung endothelial cell lysates from 8-week-old WT and CRIF1 knockout mice were collected for homogenization by RIPA buffer obtained from Cell Signaling. Then, 20 μg of the homogenates were used in the Western blotting experiments.

Following the treatments, cells were washed twice with cold PBS and lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM tetrasodium phosphate, 40 mM β-glycerophosphate, 2 mM ethylenediaminetetraacetate (EDTA), 2 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetate (EGTA), 1% Triton X, 10% glycerol, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate and protease inhibitors. The cell lysates were transferred to 1.5-ml Eppendorf tubes, homogenized and centrifuged at 10,000 × g for 5 min. at 4°C. The supernatant was transferred to a fresh tube, and protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad). Equal amounts of protein samples were resolved on SDS-PAGE and transferred onto Amersham Hybond ECL 0.45 μM nitrocellulose membranes (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The membranes were immunoblotted with actin, AMPK, P-AMPKα^Thr172, Bcl-2, caspase 3 full length, eNOS, P-eNOS (Cell Signaling, Danvers, MA, USA), AT1R, AT2R or p53 (Santa Cruz, Dallas, TX, USA) antibodies, followed by secondary antibodies. The signals were visualized using Thermo Scientific Pierce ECL Western Blotting Detection reagents (Thermo Fisher Scientific) at VersaDoc 3000 Gel Imaging System (Bio-Rad).

Western blot was performed as described in our previous publications [21 (link)]. In brief, the myocardial tissues were lysed with ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (Sigma-Aldrich, MO, USA). After protein concentration measurement by the modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Then, they were probed with antibodies against cleaved caspase-3, gp91^phox, iNOS, p-eNOS, eNOS, Notch1 ICD, PCNA, Hes1, Pten, p-Akt, Akt, and β-actin (1 : 1000, Cell Signaling Technology, MA, USA) overnight (4°C) followed by incubation with HRP-conjugated secondary antibodies (1 : 5000 Zhongshan Biotechnology, Beijing, China) for 1 h (room temperature). The SuperSignal ECL kit (Thermo Fisher Scientific, Rockville, MD, USA) was employed to detect the antigen-antibody complexes. The bands were quantified and analyzed using an image analyzer Quantity One System (Bio-Rad, CA, USA). The results were expressed as density values normalized to β-actin.

The cells were lysed with RIPA buffer (Thermo Fisher Scientific, United States). A BCA protein assay kit (Thermo Fisher Scientific, United States) was used to detect the concentration of total proteins. Equal protein (50 μg) was loaded on a 10% SDS gel and transferred to polyvinylidene fluoride membranes. Subsequently, membranes were blocked in 5% milk in TBST for 2 h. Primary antibodies against PERK (1:2,000; Genetex, United States), XBP1 (1:2,000; Sigma, CA, United States), ATF6 (1:2,000; Sigma, CA, United States), CHOP (1:2,000; Sigma, CA, United States), Caspase-12 (1:2,000; Sigma, CA, United States), JNK (1:2,000; Sigma, CA, United States), GRP78 (1:5,000; Sigma, CA, United States), p-eNOS (1:5,000; Cell Signaling, MA, United States), CaMKII (1:5,000; Genetex, United States), P-gp (1:5,000; Sigma, CA, United States), MMP9 (1:5,000; Sigma, CA, United States), and β-actin (1:5,000; Abcam, MA, United States) were utilized to incubate membranes at 4°C overnight. Membranes were washed and incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:5,000; Proteintech, China) for 2 h. Western blot bands were analyzed by Bio-rad Image Lab (v5.2.1).