Total RNA was isolated from the heart tissue by the use of Trizol reagent (Invitrogen). Relative quantification by real-time PCR involving SYBR Green was performed by ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The oligonucleotide primer sequences were showed in S1 Table . GAPDH RNA was amplified as an internal control. The extract proteins were separated by 8–10% SDS-PAGE, then transferred to nitrocellulose membranes. The membranes were probed with the antibodies including anti-phospho-AMPK (Thr 172), anti-AMPK (Cell Signaling Technology), anti-NOX4 (Abcam), anti-NOX2, anti-SOD1, anti-SOD2, anti-CAT, anti-eukaryotic translation initiation factor 5 (anti-eIF5) or anti-GAPDH (Santa Cruz Biotechnology).
Anti eif 5
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-eIF-5 is a primary antibody that recognizes the eukaryotic translation initiation factor 5 (eIF-5) protein. eIF-5 is a GTPase-activating protein that plays a key role in the initiation of protein synthesis by facilitating the recruitment of the 40S ribosomal subunit to the mRNA.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (2 mentions)
Science imaging system, by Bio-Rad (2 mentions)
Anti nox4, by Abcam (1 mentions)
Anti nox2, by Abcam (1 mentions)
Anti sod1, by Santa Cruz Biotechnology (1 mentions)
Abi prism 7700 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Anti sod2, by Santa Cruz Biotechnology (1 mentions)
Anti cat, by Santa Cruz Biotechnology (1 mentions)
Anti phospho ampk, by Cell Signaling Technology (1 mentions)
Anti ampk, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti mfn2, by Santa Cruz Biotechnology (1 mentions)
Anti total erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Applygen (1 mentions)
Anti h3, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
5 protocols using anti eif 5
1
Cardiac Tissue Total RNA Extraction and Gene Expression Analysis
2
Western Blot Analysis of Mfn2 and ERK1/2
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Total protein was extracted in RIPA buffer (CxBio, Shanghai, China) supplemented with phenylmethanesulfonyl (PMSF, CxBio). The protein concentrations were measured using the BCA Protein Assay (Applygen, Beijing, China). The samples were separated on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane, which was blocked in 5% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. After the washing steps, the membranes were incubated with horseradish peroxidase-labelled secondary antibodies for 1 h at room temperature. The bands were visualized using a chemiluminescence detection system, and the densitometric results were analysed with Image J software. EIF5 levels were used as internal controls for protein normalization.
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
3
Western Blot Analysis of H9C2 Cell Proteins
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The protein of H9C2 cells was extracted with RIPA Lysis Buffer (Beyotime, China) containing 1% phenylmethanesulfonyl fluoride on ice. The protein concentration was detected according to bicinchoninic acid Kits. We mixed 30 μg proteins with 5× SDS-PAGE sample loading buffer. Following heating at 95°C for 5min, denatured proteins were subjected to 10% Tris-glycine gel and transferred electrophoretically to polyvinylidene difluoride membranes (Bio-Rad). Then 5% Bovine serum albumin in Tris-buffered saline with 0.1% Tween 20(TBST) was used to block non-specific sites at room temperature for 1h. The membranes were incubated with the primary antibodies overnight at 4°C and washed with TBST three times. Secondary HRP-conjugated antibodies (1:4000) were incubated with the membranes for 1 h at 37°C. Autoradiographs were quantitated by a densitometry Science Imaging system (Bio-Rad, Hercules, CA). Data were analyzed by Quantity One (Bio-Rad, USA). Following antibodies were used in this study: anti-phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA,USA), anti-phospho-PKB(Ser473) (Cell Signaling Technology), anti-H3(Cell Signaling Technology), anti-ERK1/2(Santa Cruz Technology, Delaware, CA, USA), anti-GAPDH(Santa Cruz Technology), anti-eIF-5 (Santa Cruz Technology), and anti-GATA4(Selleckchem Technology, Houston,TX, USA).
4
Western Blot Analysis of Cardiac Signaling
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The cardiac tissue cells were rinsed and homogenized in RIPA lysis buffer containing protease inhibitor PMSF. The insoluble protein lysate was removed by centrifugation at 12,000 rpm for 5 min at 4°C. 50 μg of the protein lysate was resolved using 12% SDS–polyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 200 mA for 60 min. The PVDF membranes were blocked one hour in 5% milk at room temperature and subjected to Western blot analysis. Following antibodies were used: anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-PKB (Ser473), anti-phospho-CaMKII, and anti-PKB (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2, anti-GAPDH and anti-eIF-5 (Santa Cruz Technology, Delaware, CA, USA). The sheets were analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.). Bands were visualized by use of a super western sensitivity chemiluminescence detection system (Pierce, IL). Autoradiographs were quantitated by densitometry (Science Imaging system, Bio-Rad, Hercules, CA), and the ratio was compared between the phosphorylated p-ERK1/2, p-AKT, p-CaMKII and total proteins ERK1/2, AKT, CaMKII, respectively.
5
Cardiac Protein Expression Analysis
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Total protein was extracted from frozen heart, resolved and electrotransferred as described. Antibodies used for western blot were as follows: anti-p16 (Abcam), anti-p19 (MBL International Corporation), anti-p21 (MBL International Corporation), anti-p53 (MBL International Corporation), anti-GATA4 (Santa Cruz Biotechnology), and anti-eif5 (Santa Cruz Biotechnology).
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