Taurine (#T0625), cisplatin (#P4394), and H2DCFDA (#287810) were purchased from Sigma-Aldrich. The MDA assay kit (#S0131S) was purchased from Beyotime Biotechnology (China). Two primary antibodies were used: MyoD1 (ab64159, Abcam), Myogenin (67082-1-lg, Proteintech), MHC (3405S, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), p-AKT (AP3434A, ABGENT), caspase 3 (19677-1-AP, Proteintech), GAPDH (10494-1-AP, Proteintech).
Myod1
Manufactured by Abcam
Sourced in United Kingdom
MyoD1 is a protein that plays a key role in the regulation of muscle cell differentiation. It acts as a transcription factor that activates the expression of muscle-specific genes. MyoD1 is commonly used in research applications to study muscle development and function.
Automatically generated - may contain errors
Lab products found in correlation
Desmin, by Abcam (2 mentions)
Mda assay kit, by Beyotime (1 mentions)
Taurine, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Caspase 3 19677 1 ap, by Proteintech (1 mentions)
Myogenin, by Proteintech (1 mentions)
Akt 10176 2 ap, by Proteintech (1 mentions)
Gapdh 10494 1 ap, by Proteintech (1 mentions)
Cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Integrin β3, by Santa Cruz Biotechnology (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Bacitracin, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
Donkey anti rabbit fitc, by Thermo Fisher Scientific (1 mentions)
Antibodies against akt, by Abcam (1 mentions)
Pdia1, by Abcam (1 mentions)
Pdia3, by Abcam (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
6 protocols using myod1
1
Taurine, Cisplatin, and Oxidative Stress
2
Protein Expression Analysis in Muscle Regeneration
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Antibodies against eMyHC, myogenin, Pax7, and β3 integrin were purchased from Santa Cruz (Dallas, TX, USA). Antibodies against AKT, p-AKT, Myod1, PDIA1, PDIA3, and GAPDH were purchased from Abcam (Cambridge, UK). Antibodies against mTOR and P-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against eMyHC were purchased from R&D (Minneapolis, MN, USA). The Cell Surface Protein Isolation Kit, Donkey anti-rabbit FITC, and donkey anti-mouse TRITC were purchased from Thermo Fisher Scientific (Waltham, MA USA). The ELISA kit for PDIA3 detection was obtained from R&D (Minneapolis, MN, USA). Cardiotoxin (CTX), LY294002, recombinant human Vitronectin and PDI inhibitors (16F16, EGCG, bacitracin, and PCAMA31) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3
Isolation and Analysis of C2C12 Proteins
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Intracellular proteins of C2C12 myoblasts were isolated using RIPA lysis buffer with protease inhibitor and phosphorylation protease inhibitor cocktails (Thermo Fisher). The homogenates were then sonicated for 35 s and centrifuged (11,000 g for 10 min at 4°C) to remove the debris. The supernatants were collected, and protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotechnology). Then, the denatured samples were subjected to SDS-PAGE and transferred to PVDF membranes (GE Healthcare) for immunoblotting analysis. After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, the membranes were probed with corresponding antibodies (MyoD1, Abcam, ab64159; GAPDH, ProteinTech, 10494-1-AP). Proteins were visualized by enhanced chemiluminescence using horseradish peroxidase–conjugated antibodies, and band densities were quantified by the ImageJ software.
4
Myoblast Differentiation and Tissue Characterization
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Cultured cells were washed with PBS, fixed, permeabilized with 0.3% Triton X-100 (Sigma), blocked with 1% bovine serum albumin (BSA, Sigma), and incubated overnight at 4 °C with the following primary antibodies: Pax7 (Abcam), Myod1 (Abcam), Myf5 (Abcam), Desmin (Abcam), Dystrophin (Abcam), Myogenin (Abcam), MyHC (DSHB), IL4Rα (Abcam), and PDGFRα (CST), this was followed by incubation with secondary antibodies for one hour at room temperature. The fusion index was determined as the ratio between the number of nuclei (at least two) incorporated into myotubes determined by immunodetection of MyHC and the total number. Tissue samples were sliced into serial 8-μm frozen sections. Tissue sections were fixed with cold acetone, permeabilized, and blocked as previously described, and then incubated overnight with primary antibodies against Dystrophin (Abcam), GFP (Abcam), CD206(Abcam), and INOS(Abcam) at 4 °C, followed by incubation with secondary antibodies for one hour at room temperature. Fluoroshield™ with DAPI (Sigma) was used to stain the nuclei. Immunoreactivity was visualized and imaged using a NIKANG fluorescence microscope (Olympus, Tokyo, Japan).
5
Protein Extraction and Western Blot Analysis
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The whole protein extraction kit (KGP2100, KeyGEN, China) was used to extract protein lysates of C2C12 cells. After being separated by electrophoresis on 10% SDS–PAGE at 80 V for 40 minutes and 120 V for 60 minutes, protein samples were transferred to PVDF membranes (Millipore), followed by the process of blocking. PVDF membranes were then incubated with diluted primary (1:1000) and secondary (1:10000) antibodies, respectively. Finally, after several washings, protein expression was determined with the NcmECL High kit (NCM Biotech) and imaged by a Tanon 5200 Multi Scanning System (Tanon). Primary antibodies were myosin heavy chain (MyHC; Abclonal), myogenin (MYOG; Abcam), myoblast determination protein 1 (MyoD1; Abcam), and α-Tublin (CST). All secondary antibodies were from AiFang biological (AFSA001, AFSA004).
6
Comprehensive Tissue Analysis Protocol
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The explanted grafts were fixed in 10% formalin and embedded in paraffin. Sections cut from the paraffin-embedded samples were subjected to H&E staining and Masson trichrome staining as well as IHC staining for CD31, CD68, MyoD1, and desmin (all 1:200; Abcam); CD80 and CD163 (both 1:200; AbD Serotec); and human nuclear antigen (HuNu; 1:200; Millipore, Bedford, MA). The slides were imaged and analyzed using the Vectra multispectral slide analysis system. The details of using the ABC kit for IHC staining are described in section 2.2.6 .
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