Ab184154

The expressions of related proteins in HUVECs were measures by WB assays. Total proteins of cells were separated by the RIPA buffer (Solarbio, Beijing, China), and Bicinchoninic Protein Assay kit (BCA, Pierce, Rockford, IL, USA) was used for quantitation. 30 μg of total protein was isolated using the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China), and moved onto the polyvinylidene fluoride (PVDF) membranes. Then, 5% non-fat dried milk was used to seal the membranes for 2 h. Primary antibodies including p-eNOS (1：2000, ab184154, Abcam, USA), p-eNOS (1：1000, ab138430, Abcam, USA), eNOS (1：1000, ab76198, Abcam, USA)，p-Akt (1：1000, #9271, Cell Signalling Technology, USA) and Akt (1：1000, #9272, Cell Signalling Technology, USA) were subsequently used to incubated the membranes overnight at 4℃. GAPDH (1：1000, ab8245, Abcam, USA) served as the internal control. Subsequently, the homologous secondary antibodies goat anti-rabbit IgG H&L (HRP; 1：7000, ab97051, Abcam, USA) and goat anti-mouse IgG H&L (HRP; 1：1000, ab150113, Abcam, USA) were added for another 1 h at room temperature. The bands were developed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA).

Cardiac tissue samples for western blotting were obtained from the infarcted area. Proteins isolated from sI/R-treated CMECs were also analyzed through western blots. In brief, proteins were separated by SDS electrophoresis and transferred to membranes using standard protocols,⁶⁷ (link) after which they were probed with antibodies against p-eNOS (Ser1117) (1:1,000, #ab184154; Abcam), ET-1 (1:1,000, #ab2786; Abcam), ICAM1 (1:1,000, #ab119871; Abcam), VCAM1 (1:1,000, #ab134047; Abcam), Gr1 (1:1,000, #ab25377; Abcam), FUNDC1 (1:1,000, #ab224722; Abcam), ATG5 (1:1,000, #12994, Cell Signaling Technology), and Beclin1 (1:1,000, #3738; Cell Signaling Technology). The blots were visualized by chemiluminescence (ECL, Immunobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, MA, USA), and the signals were quantified by densitometry. GAPDH (analyzed with an antibody from Santa Cruz Biotechnology) served as a loading control.⁶⁸ (link)

Cells were lysed using 200 µl lysis buffer NP40 (Beyotime Institute of Biotechnology). The concentration of the samples was determined by BCA protein quantitative kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated using 10% SDS-PAGE gels and transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA). After being blocked with 5% non-skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies as listed: cleaved caspase-3 (ab32042, 1:500; Abcam, Cambridge, UK), Fas (ab82419, 1:1,000), Fasl (ab15285, 1 ug/ml), Bax (ab32503,1:2,000), Bcl-2 (ab32124, 1:1,000), p-AKT (ab812831:5,000), AKT 1/2 (ab182729, 1:5,000), p-eNOS (ab184154, 1:1,000), eNOS (ab76198, 1:1,000), and GAPDH (ab8245, 1:5,000), at 4°C overnight. Then, horseradish peroxidase-conjugated secondary antibody (ab6721, 1:4,000) was added and maintained at room temperature for 1 h. The blot bands were developed using BeyoECL Star (Beyotime Institute of Biotechnology). The gray density was calculated with Quantity One software version 4.6 (Bio-Rad Laboratories, Inc.).

Reperfused hearts tissues and cultured CMECs were washed twice with ice-cold PBS and harvested in RIPA Lysis and Extraction Buffer (Life Technologies), Halt Protease Inhibitor Cocktail (Life Technologies), and Halt Phosphatase Inhibitor Cocktail (Life Technologies). A Pierce BCA Protein Assay Kit (Life Technologies) was used for the colorimetric detection and quantitation of total protein [43 (link)]. A total of 25 μg of protein was separated on 4%–12% Tris-Glycine Mini Gels (Life Technologies) and transferred to PVDF membranes (Merck Millipore). The membranes were probed with antibodies to SERCA (1:1111, Abcam, #ab150435), MCU (1:1000, Cell Signaling Technology, #14997), p-eNOS (Ser1117) (1:1000, Abcam, #ab184154), ET-1 (1:1000, Abcam, #ab2786), and GAPDH (1:5000) (MilliporeSigma, cat# ABS16). Protein signals were detected using horseradish peroxidase (HRP)–conjugated secondary antibodies and enhanced chemiluminescence (ECL) western blotting detection reagents (Thermo Fisher Scientific, MA, USA) [44 (link)].

The protein was extracted from the cells with the RIPA (Beyotime, P0013C). The protein concentration was determined using the bicinchoninic acid (BCA) method, and then the protein was incubated at 99°C for 10 minutes to denature the protein. After that the samples were separated by the 10% sodium dodecyl sulfate (SDS) gels (Beyotime, P0012A). Then the proteins were transferred to the PVDF (polyvinylidene fluoride) membranes (Millipore, USA). After that the targeted proteins were incubated with the primary anti-body p-eNOS (Abcam, ab76199), eNOS (Abcam, ab184154), endothelin-1 (ET-1) (Abcam, ab2786), Bax (CST, #14796), Bcl-2 (CST, #15071), cleaved caspase-3 (CST, #9664), caspase-3 (CST, #9662), α1-AMPK (Abcam, ab32047), Nox-2 (Abcam, ab80508), and GAPDH (CST, #5174) at 4°C overnight. In the second day, the membranes were washed by the PBST (phosphate-buffered saline Tris) for 3 times. After that, the membranes were incubated with the second anti-body rabbit IgG (CST, #14708) and anti-mouse IgG (CST, #7076) for 2 hours at the room temperature. Then the membranes were washed by the PBST again. Then the membranes were exposed in the machine.