We analysed the expression of cardiac lipid metabolism proteins by the Western blot method as previously described in detail.28 As severe LV hypertrophy is related to the impaired angiogenesis pathway, we also studied the expression of key proteins that regulate angiogenesis and signal hypoxia. Primary antibodies: HIF1α (ab463, Abcam; 1:500); fatty acid transporter (CD36; ab133625, Abcam; 1:1000); carnitine palmitoyltransferase 1 (CPT1; NBP1‐59576, Novus Biological; 1:1000); fatty acid‐binding protein (FABP3; ab133585, Abcam; 1:1000); acyl‐CoA dehydrogenase (ACADL; ab196655, Abcam; 1:3000); medium‐chain acyl‐CoA dehydrogenase (MCAD; ab110296, Abcam; 1:1000); 2,4‐dienoyl‐CoA reductase (DECR1; ab198848, Abcam; 1:2000). AMP‐activated protein kinase (AMPK; 2532 s, Cell Signaling; 1:1000); phosphorylated AMPK (Thr 172) (pAMPK; 2535 s, Cell Signaling; 1:500); SIRT1 (8469 s, Cell Signaling; 1:1000); PPARα (ab8934, Abcam; 1:1000); retinoid X receptor (RXRα; 3085 s, Cell; 1:1000); peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α; 20,658‐1‐AP, Proteintech; 1:1000). Targeted bands were normalized to the expression of cardiac GAPDH (sc‐32,233, Santa Cruz Biotechnology; 1:2000).
Ab196655
Manufactured by Abcam
Sourced in United States
Ab196655 is a laboratory equipment product. It is a key component for conducting scientific experiments and research. The core function of this product is to facilitate specific tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab128568, by Abcam (3 mentions)
Ab133625, by Abcam (2 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Pgc 1α, by Proteintech (1 mentions)
Gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Ab463, by Abcam (1 mentions)
Ab8934, by Abcam (1 mentions)
Ab133585, by Abcam (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab57602, by Abcam (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ab92461, by Abcam (1 mentions)
Ab56889, by Abcam (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Anti rabbit 7074, by Cell Signaling Technology (1 mentions)
Ab192306, by Abcam (1 mentions)
Ab126706, by Abcam (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
T6074, by Merck Group (1 mentions)
4 protocols using ab196655
1
Cardiac Lipid Metabolism and Angiogenesis
2
Mitochondrial Protein Expression Analysis
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Fibroblasts grown in T75 flasks at 90–95% confluence were harvested by trypsinization, pelleted by centrifugation and stored at -80 °C. The BCA Protein Assay (Thermo scientific) quantified protein concentration. Proteins were separated on NUPAGE gels (Invitrogen) and transferred to nitrocellulose membranes. After 1 h of blocking, membranes were incubated with primary antibodies. The primary antibodies used as follows: VLCAD (GeneTex, Rb pAb GTX114232) 1:1000, MCAD (Abcam, Rb mAb ab92461) 1:1000, LCAD (Abcam, Rb mAb ab196655) 1:1000, TFPa (Abcam, Rb pAb ab54477) 1:500, TFPb (Bethyl, Rb A305-020A) 1:3000, CPT1α (Abcam, Ms mAb ab128568) 1:1000, MFN1 (Abcam, Ms mAb ab57602) 1:400, MFN2 (Abcam, Ms mAb ab56889) 1:400, DRP1 (Abcam, Rb mAb ab184247) 1:100 and GAPDH (Cell Signaling Technology, Rb mAb 2118S) 1:30000 dilutions overnight at 4 °C. The membranes were then incubated with fluorescent conjugated secondary antibodies for 1 h. The secondary antibodies used and their concentration as follows: Antibody DyLight 800 conjugated Anti-Rabbit IgG made in goat (611-145-002), Antibody DyLight 680 conjugated Anti-Rabbit IgG made in goat (611-144-003), Antibody DyLight 800 conjugated Anti-Mouse IgG made in goat (610-145-002), and Antibody DyLight 680 conjugated Anti-Mouse IgG made in donkey (610-744-124). Band signal intensities were obtained using Odyssey imaging system.
3
Western Blot Analysis of Metabolic Proteins
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Primary hepatocytes, liver and intestine tissues were collected and lysed in RIPA buffer containing PMSF and phosphatase inhibitor cocktail for 10 min on ice. After determining protein concentrations, we boiled the lysates and subjected them to SDS-PAGE. Subsequently, proteins were transferred onto polyvinylidene fluoride membranes, which were blocked with 5% defatted milk for 1 h at room temperature. The membrane was incubated overnight at 4 °C with primary antibodies and then incubated with secondary antibodies for 1 h at room temperature. Immunoreactive signals were detected with ECL reagent (Thermo Scientific). Image J was used to quantify the Western bands.
Antibodies against SCD1 (2794), FABP1 (13368), CS (14309), COX IV (11967), AMPKα (5832), p-AMPKα (Thr172, 2535), ACC (3662), p-ACC (Ser79, 3661), GAPDH (5174), β-actin (3700), HSP90 (4877), anti-mouse (7076) and anti-rabbit (7074) secondary antibodies were obtained from Cell Signaling Technology. Antibodies to CPT1A (ab128568), ACADL (ab196655), FADS1 (ab126706), mt-ND3 (ab192306), CD36 (ab133625) and GRIM19 (ab110240) were purchased from Abcam. Antibody against Ndufs4 (WH0004724M1) was from Sigma. Antibody to mt-ND2 (LS-C498022) was acquired from Lifespan.
Antibodies against SCD1 (2794), FABP1 (13368), CS (14309), COX IV (11967), AMPKα (5832), p-AMPKα (Thr172, 2535), ACC (3662), p-ACC (Ser79, 3661), GAPDH (5174), β-actin (3700), HSP90 (4877), anti-mouse (7076) and anti-rabbit (7074) secondary antibodies were obtained from Cell Signaling Technology. Antibodies to CPT1A (ab128568), ACADL (ab196655), FADS1 (ab126706), mt-ND3 (ab192306), CD36 (ab133625) and GRIM19 (ab110240) were purchased from Abcam. Antibody against Ndufs4 (WH0004724M1) was from Sigma. Antibody to mt-ND2 (LS-C498022) was acquired from Lifespan.
4
Protein Expression Analysis in Kidney Tissue
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Cells were lysed in 1 × SDS sample buffer. Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments) in RIPA lysis buffer on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% wt/vol. SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-PPARα (ab24509, Abcam, Cambridge, MA, USA), anti-CPT1α (ab128568, Abcam), anti-ACADL (ab196655, Abcam), anti-HK2 (ab76959, Abcam), anti-LDH (3558, Cell Signaling Technology, Danvers, MA, USA), anti-PDK1 (ab110025, Abcam), anti-HIF-1α (ab1, Abcam), anti-PHD2 (4835, Cell Signaling Technology), and anti-tubulin (T6074, Sigma Aldrich). Western blot was performed at least three times independently. Chemiluminescence is applied for detecting proteins on western blot membranes. The enhanced chemiluminescent ECL substrate (32209, Thermofisher Scientific, Carlsbad, CA, USA) enables immunodetection of horseradish peroxidase (HRP)-conjugated secondary antibodies using an imaging system. Quantification was performed by measurement of the intensity of the signals with the aid of Image J software package.
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