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).
Gapdh sc 32233
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
GAPDH (sc-32233) is a highly specific antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in the glycolytic pathway, catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. This antibody can be used to detect and quantify GAPDH expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (3 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
β catenin 71 2700, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Smad4 ab 40759, by Abcam (2 mentions)
Pi3k p110α, by Cell Signaling Technology (2 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (2 mentions)
α tubulin sc 5286, by Santa Cruz Biotechnology (2 mentions)
Pras40, by Cell Signaling Technology (2 mentions)
Aktpos473, by Cell Signaling Technology (2 mentions)
Aktpos473 4, by Cell Signaling Technology (2 mentions)
Akt2pos474, by Cell Signaling Technology (2 mentions)
Aktpot308, by Cell Signaling Technology (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fus tls sc 47711, by Santa Cruz Biotechnology (2 mentions)
82 protocols using gapdh sc 32233
1
Cardiac Lipid Metabolism and Angiogenesis
2
Comprehensive Antibody Validation for GLI2 and Hedgehog Pathway
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Antibodies against GLI2 H-300 (sc-28674) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology; GLI3 (AF3690) from R and D Systems; SHH (ab53281), SMO (ab38686) and S100A2 (ab109494) from Abcam; GLI2 (18989–1-AP), KRAS (12063–1-AP), Tubulin (66031–1-Ig), E-Cadherin (20874–1-AP), and ESRP1 (21045–1-AP) from Proteintech; GLI1 (2534), FLAG (2368), ZEB1 (3396), Vimentin (5741), YAP1 (4912), N-Cadherin (4061), GATA6 (5851) from Cell Signaling Technology; Keratin 14 (905304) and Keratin 5 (905504) from BioLegend; Peroxidase goat anti-rabbit (PI-1000), horse anti-goat (PI-9500), and horse anti-mouse IgG (PI-2000) antibodies from Vector Labs. Keratinocyte SFM and supplements, RPM1, DMEM, DMEM F12 (1:1), fetal bovine serum (FBS) were purchased from Corning; tet system approved FBS from Clontech. Silencer select siRNA sequences against human GLI2, GLI1 and KRAS were purchased from Ambion.
3
Protein Expression Analysis Protocol
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Cell lysates were prepared using RIPA buffer containing a protease and phosphatase inhibitor cocktail (Calbiochem, La Jolla, CA, USA). The total protein was quantified using Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 μg per lane) from the samples were separated by 8% or 10% SDS-PAGE and probed with the indicated antibodies. Antibodies against survivin (#2808), ERK (#9102), pERK (#9106), JNK (#9252), pJNK (#9251), p38 (#9212), and pp38 (#9216) were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against BCl (#sc-783), Bax (#sc-493), caspase-3 (#sc-7148), caspase-9 (#sc-7885), PARP (#sc-7150), β-actin (#sc-47778) and GAPDH (#sc-32233) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary horseradish peroxidase-conjugated antibodies used were goat anti‑rabbit IgG and goat anti‑mouse IgG (SC‑2004 and SC-2005, respectively; Santa Cruz Biotechnology, Dallas, TX, USA). Relative intensities of the bands were analyzed with a GS‑710 Image Densitometer (Bio‑Rad Laboratories). Results are representative of at least five independent experiments.
4
CDK9 Inhibitor LDC000067 Protocol
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The CDK9 inhibitor, LDC000067, was purchased from Selleck (Houston, TX). LDC000067 was dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments and in CMC-Na (1%) for in vivo study. Atorvastatin was purchased from Pfizer Ireland Pharmaceuticals (New York, USA). Antibodies against NF-κB p65 (sc-8008), Lamin B (sc-56144) and GAPDH (sc-32233) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against CDK9 (2316S), p-CDK9 (2549S) and IκB (4814S) were from Cell Signaling Technology (Danvers, MA); antibodies against α-SMA (ab32575), Vimentin (ab8978), OPN (ab8448), PCNA (ab29), β-actin (ab8227) and CD68 (ab955) were from Abcam (Cambridge, MA, USA). Mouse CDK9 ELISA kit (E-EL-M0378c) was from Elabscience Biotechnology Co., Ltd (Wuhan, China).
5
Antibody-based Protein Analysis Protocol
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Antibodies against E2F1 (sc-251), DDR1 (sc-532), E-cadherin (sc-8426), vimentin (sc-6260), MMP2 (sc-10736), MMP9 (sc-10737), and GAPDH (sc-32233) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The p-AKT (S473) (#4051), AKT (#2966), p-STAT3 (Y705) (#4113), and STAT3 (#9139) antibodies were from Cell Signaling Technology (Beverly, MA, USA). The siRNAs targeting STAT3 were from Santa Cruz.
6
Muscle Atrophy Protein Expression Analysis
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GCM and EDL muscles were homogenized in RIPA buffer containing protease/phosphatase inhibitor cocktails and then centrifuged at 12,000 rpm for 1 h at 4°C (Brock Symons et al., 2023 (link)). Proteins (30 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure their expression. The separated proteins were transferred to a nitrocellulose membrane and then incubated overnight at 4°C with primary antibodies against muscle atrophy, viz. F-box (MAFbx/atrogin-1, ab-168372, 1:1000, Abcam, Cambridge, UK), MuRF1 (ab-172479, 1:1000, Abcam), myosin heavy chain (MyHC, sc-376157, Santa Cruz Biotechnology, CA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH. Sc-32233; Santa Cruz Biotechnology). After incubation and rinsing with T-BST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 h at room temperature (25°C). Membranes were analyzed using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of Stem Cell Regulators
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Cells and tumors were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, and Halt protease inhibitor cocktail [Pierce]) and total protein was quantified using Bio-Rad protein assay reagent. An equal amount of protein was loaded on 10% or 15% SDS-PAGE gels and immunoblotted. STRA6 antibody was generated in the Lerner Research Institute Molecular Biotechnology core as previously described (Berry et al., 2013 (link), Bouillet et al., 1997 (link)). RBP4 antibody was purchased from Atlas Antibodies (HPA001641) and serum RBP4 was blotted using antibody from Dako (#A0040). Antibodies to NANOG (#4903), SOX2 (#3579), STAT3 (#4904), and phospho-STAT3 (Y705) (#9145) were from Cell Signaling Technology. RARβ (SC-552), βActin (SC-47778), and GAPDH (SC-32233) antibodies were from Santa Cruz Biotechnology.
8
Western Blot Analysis of Mouse Kidney
9
Western Blot Analysis of Kidney Proteins
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Kidney cortex extracts were homogenized in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitor cocktail and PMSF (Sigma-Aldrich). Protein content was determined by the Folin-phenol method,65 (link) using BSA as a standard. Aliquots of 30 μg proteins were run on 10%–12% SDS-PAGE under reducing conditions, and western blotting was carried out as previously described.65 (link) The following primary antibodies were used: GAPDH, sc-32233 (1:1,000, Santa Cruz); α-SMA, MAB1420 (1:1,000, R&D Systems, Minneapolis, MN, USA); Desmin, ab32362 (1:1,000, Abcam); B cell lymphoma 2 (Bcl2), 2876 (1:1,000, Cell Signaling Technology, Danvers, MA, USA); and Bcl-2-associated X (Bax), 2772S (1:500, Cell Signaling Technology). The secondary antibodies were anti-rabbit and anti-mouse IgG-horseradish peroxidase (HRP) (NA934-1 and NA931, 1:5,000 and 1:10,000, GE Healthcare, Buckinghamshire, UK). Proteins were detected by chemiluminescence using the ECL system (GE Healthcare) and ChemiDoc XRS+ (Bio-Rad). Quantification of western blots was performed with ImageJ software (NIH).
10
Melanogenesis Regulation by TCQA and α-MSH
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HEM and B16F10 were seeded at density of 5 × 105 cells per 100 mm petri dish and then treated with 0, 10, and 25 μM of TCQA, and 200 nM of α-MSH. After 4, 8, 12, 24, and 48 h, total protein extraction was achieved using radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, United States) and protease inhibitor following the manufacturer’s instructions.
The quantification was assessed using a 2-D Quant kit according to manufacturer’s instructions (GE Healthcare, Chicago, United States).
The protein sample (15 μg/well) was separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, NJ, United States). After being blocked, the membranes were incubated with primary antibodies against β-catenin 71–2700 (Thermo Fisher Scientific, MA, United States), Mitf (Abcam, Rockford, United States), tyrosinase (Abcam, Rockford, United States), and Tyrp1 sc-166857, Dct sc-74439, and GAPDH sc32233 (Santa Cruz Biotechnology, TX, United States). After overnight incubation at 4°C, the second antibody goat anti-rabbit IRDye 800 CW or IRDye 680 LT goat anti-mouse was added. Then the expression was detected using LI-COR Odyssey Infrared Imaging System (LI-COR, NE, United States).
The quantification was assessed using a 2-D Quant kit according to manufacturer’s instructions (GE Healthcare, Chicago, United States).
The protein sample (15 μg/well) was separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, NJ, United States). After being blocked, the membranes were incubated with primary antibodies against β-catenin 71–2700 (Thermo Fisher Scientific, MA, United States), Mitf (Abcam, Rockford, United States), tyrosinase (Abcam, Rockford, United States), and Tyrp1 sc-166857, Dct sc-74439, and GAPDH sc32233 (Santa Cruz Biotechnology, TX, United States). After overnight incubation at 4°C, the second antibody goat anti-rabbit IRDye 800 CW or IRDye 680 LT goat anti-mouse was added. Then the expression was detected using LI-COR Odyssey Infrared Imaging System (LI-COR, NE, United States).
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