Myocardial tissues were ground into homogenate according to the mass volume ratio 1:10 (mg/μL) adding 1 × tissue lysate, and then the homogenate was centrifuged for 10 min at 4°C for supernatant. The supernatant was collected and added into the same volume of 2 × loading buffer at 100°C for 5 min. Each channel was added with equal amount of protein mixture for polyacrylamide gel electrophoresis, transferred into membrane. The primary antibodies including α-myosin heavy chain (α-MHC; 1:200, Santa Cruz Biotechnology, USA), β-MHC (1:200, Santa Cruz Biotechnology, USA), CBS (1:2000, Santa Cruz Biotechnology, USA), CSE (1:500, Sigma, USA), MPST (1:4000, Santa Cruz Biotechnology, USA), superoxide dismutase 1 (SOD1; 1:5000, Stressgen, USA), SOD2 (1:5000, Stressgen, USA), β-actin (1:2000, Santa Cruz Biotechnology, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000, Kangcheng, Shanghai, China) were added into membrane overnight at 4°C for incubation, and then washed by TTBS for 10 min/time of total of four times, with secondary antibodies for 1 h at room temperature for full incubation. AlphaImager gel imaging system was used to scan the protein band and measure the optical density of the protein band, and β-actin and GAPDH as internal reference protein were used to correct (Luo et al., 2013 (link)).
β mhc
Manufactured by Santa Cruz Biotechnology
Sourced in United States
β-MHC is a protein that serves as a key component of the myosin heavy chain, which is essential for muscle contraction. It plays a crucial role in the structure and function of the sarcomere, the basic unit of skeletal and cardiac muscle.
Automatically generated - may contain errors
Lab products found in correlation
Total erk1 2, by Cell Signaling Technology (3 mentions)
Total mek1 2, by Cell Signaling Technology (3 mentions)
Lamin b, by Santa Cruz Biotechnology (2 mentions)
Phospho jnk1 2 thr183 tyr185, by Cell Signaling Technology (2 mentions)
Total p38, by Cell Signaling Technology (2 mentions)
Total jnk1 2, by Cell Signaling Technology (2 mentions)
Cell culture reagents, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phototope hrp western blot detection system, by Cell Signaling Technology (1 mentions)
Mef2c, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat igg, by R&D Systems (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Bio-Rad (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Vegfr2, by Santa Cruz Biotechnology (1 mentions)
P vegfr2, by Santa Cruz Biotechnology (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
9 protocols using β mhc
1
Western Blot Analysis of Myocardial Proteins
2
Antibody Procurement for Protein Analysis
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Antibodies against the following proteins were purchased from Santa Cruz Biotechnology: atrial natriuretic peptide (sc‐20158), β‐MHC (myosin heavy chain beta; sc‐53090), and Lamin B (sc‐6217). Antibody against GAPDH was obtained from Bioworld Technology (MB001). Antibodies against FLAG were obtained from Sigma‐Aldrich (F3165). Antibodies against HA were purchased from Cell Signaling Technology (#3724). Antibodies against NULP1 were obtained from Santa Cruz (sc‐514203) and BETHYL (A303‐087A).
3
Cardiomyocyte Differentiation Markers in MSCs
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Cardiomyocyte-specific markers (GATA-4, Nkx2.5, β-MHC and MEF2c) of the induced MSCs were also identified with immunoblotting every week. Total protein was extracted from MSCs and differentiated cardiomyocyte-like cells, and then quantified with a BCA protein assay kit (Pierce, Rockford, IL, USA). Cell lysates were separated by SDS-PAGE (10%) and incubated with goat anti-pig polyclonal primary antibodies (anti-GAT4, -Nkx2.5, -β-MHC and -MEF2c; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and donkey anti-goat IgG (R&D) secondary antibody conjugated with horseradish peroxidase. GAPDH was used as a loading control. Complexes were detected by chemiluminescence (Phototope-HRP Western Blot Detection System; Cell Signaling, Danvers, MA, USA).
4
Protein Expression Analysis in Left Ventricular Tissue
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Briefly, total protein of left ventricular tissue was extracted using RIPA Buffer (BioRad), which contained Protease Inhibitor Cocktail (Sigma‐Aldrich). BCA method was used to detect the concentration. The sample was fractionated and transferred, then blocked and incubated with ANP, β‐MHC, bax, bcl‐2, NOX2, NOX4, VEGF, VEGFR2, p‐VEGFR2 and STAT3 antibody (diluted 1:200, Santa Cruz, USA), and then incubated with a horseradish peroxidase‐conjugated goat mouse antibody (diluted 1:2,000, Santa Cruz, USA). The relative level of protein was calculated by normalizing mean ray value to β‐actin. A chemiluminescence system (ChemiDoc XRS, BioRad) was used to detect protein bands.
5
Antibody Immunoblotting for Mitochondrial Proteins
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Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX): ANP (sc20158 1:200), β‐MHC (sc53090 1:200), TIM50 (sc55338 1:200), and ASK1 (sc7931, 1:200). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): GAPDH (#2118 1:1000), phospho‐ASK1Thr845 (3765, 1:1000), phospho‐MEK1/2Ser217/221 (#9154 1:1000), total‐MEK1/2 (#9122 1:1000), phospho‐ERK1/2Thr202/204 (#4370 1:1000), total‐ERK1/2 (#4695 1:1000), phospho‐JNK1/2Thr183/Tyr185 (#4668 1:1000), total‐JNK1/2 (#9258 1:1000), phospho‐P38Thr180/182 (#4511 1:1000), and total‐P38 (#9212 1:1000). Drp1 (#184272, 1:500), Mfn1 (#57602, 1:500), Mfn2 (#56889, 1:500) and Nrf2 (#31163, 1:500) were purchased from Abcam (Cambridge, MA), Fetal calf serum was purchased from HyClone (Waltham, MA). The other reagents for cell culture were purchased from Sigma (St. Louis, MO).
6
Western Blot Analysis of Signaling Proteins
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Antibodies specific to the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA) and used in western blot experiments: phospho‐MEK1/2Ser32/36 (9154, 1:1000 dilution); total‐MEK1/2 (9122, 1:1000 dilution); phospho‐ERK1/2Thr202/Tyr204 (4370, 1:1000 dilution); total‐ERK1/2 (4695, 1:1000 dilution); phospho‐JNK1/2Thr183/Tyr185 (4668, 1:1000 dilution); total‐JNK (9258, 1:1000 dilution); phospho‐P38Thr180/Tyr182 (4511, 1:1000 dilution); and total‐P38 (9212, 1:1000 dilution). Antibodies specific to the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX): ANP (sc20158, 1:200 dilution) and β‐MHC (sc53090, 1:200 dilution). Anti‐GAPDH (MB001, 1:10000 dilution) was purchased from Bioworld Technology (Harrogate, UK). Anti‐ACE3 (BAM‐73‐006‐EX, 1:500 dilution) was purchased from COSMO BIO Co, Ltd (Tokyo, Japan). The BCA protein assay kit was purchased from Pierce (Rockford, IL). Peroxidase‐conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and used for visualization. Unless otherwise noted, cell culture reagents and all other reagents were obtained from Sigma (St. Louis, MO).
7
Betulinic Acid's Bioactive Effects
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Betulinic acid was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and penicillin-streptomycin F-actin, Alexa Four 488 phalloidin, dichlorofluorescin diacetate (DCFH-DA), DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Doxorubicin, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT), primary antibodies for p38, ERK, JNK, p-ERK1/2, p-p38, NfκB p65, Bcl-2, Bax, β-actin (sc-47778), β-MHC, MLC-2v, p-GATA-4, GATA-4, Calcineurin, HDAC, LaminB, and HRP conjugated secondary antibodies raised against mouse, rabbit and goat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NFAT-c3, Caspase-3, Caspase-9, Bax, and α-tubulin were purchased from Cell signaling technology (Danvers, MA, USA). ANP and BNP were purchased from ABCam (Danvers, MA, USA).
8
Antibody Source and Reagent Details
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The antibodies against ANP (atrial natriuretic peptide) (sc20158) and β-MHC (β-myosin heavy chain) (sc53090) were from Santa Cruz Biotechnology. The antibody against TRIM32-(28–984) was from ProSci. The antibody against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (MB001) was from Bioworld Technology. The antibodies against p-MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (9154), total MEK1/2 (9122), p-ERK1/2 (4370), total ERK1/2 (4695), p-JNK1/2 (c-Jun N-terminal kinase 1/2) (4668), total JNK1/2 (9258), p-p38 (4511), total p38 (9212), p-Akt (4060), total Akt (4691), p-GSK3β (glycogen synthase kinase 3β) (9322), total GSK3β (9315), p-mTOR (mammalian target of rapamycin) (2971), total mTOR (2983) and total p70 (2708) were from Cell Signaling Technology. The antibody against p-p70 was from GeneTex (GTX50304). The BCA protein assay kit was from Pierce. FBS was from Hyclone. Cell culture reagents and all other reagents were from Sigma.
9
Immunohistochemical Analysis of Cardiac Proteins
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The paraffin sections were manufactured as described previously. α-MHC, β-MHC and Foxp3 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a dilution of 1:200 according to the manufacturer’s instruction. To visualize this reaction after incubation with a secondary antibody at room temperature, the slides were incubated with 3,3 N-diaminobenzidine tetrahydrochloride (DAB). The reaction was stopped by immersing the slides in distilled water, and then the sections were counterstained with haematoxylin, mounted and examined. The OD values were calculated by Image Pro-Plus5.1 (Media Cybernetics, Silver Spring, MD, USA).
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