myhc rabbit polyclonal antibody  (Proteintech)

Journal: Poultry science

Article Title: IGFBP7 promotes the proliferation and differentiation of primary myoblasts and intramuscular preadipocytes in chicken.

doi: 10.1016/j.psj.2024.104258

Figure Lengend Snippet: Figure 3. IGFBP7 promotes CPMs differentiation. (A, B) Relative IGFBP7 mRNA expression after transfection with the listed nucleic acids. (C, D) Relative MyoD, MyoG, MyHC, and Myomaker mRNA expression. (E, G) MyHC Immunofluorescence staining of CPMs. (F, H) Myotube area (%) of CPMs. (I, J) The protein expression of MyHC was determined by Western blot assay in CPMs transfected with pcDNA3.1-IGFBP7 or pcDNA3.1. (K, L) The protein expression of MyHC was determined by Western blot assay in CPMs transfected with si-IGFBP7 or si-NC.

Article Snippet: The antibodies used for Western blot analysis include MyHC mouse monoclonal antibody (B103; DHSB, Lowa City, IA, USA, 1:200), IGFBP7 antibody (CSB-PA16259A0Rb; CUSABIO, Wuhan, China, 1:1000), GAPDH antibody (60004-1-Ig; Proteintech, Wuhan, China, 1:20000), and Beta Actin antibody (66009-1-Ig; Proteintech, Wuhan, China, 1:20000) as primary antibodies.

Techniques: Expressing, Transfection, Staining, Western Blot

Journal: International journal of molecular sciences

Article Title: miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts.

doi: 10.3390/ijms23179545

Figure Lengend Snippet: Figure 2. miRNA-10a-5p inhibits chicken myoblasts’ differentiation. (A,B) The qRT-PCR results of differentiation marker genes MYOD1 and MYOG after transfection with miRNA-10a-5p mimic and inhibitor. (C,D) The protein expression of MYHC and MYOD1 after transfection with miRNA- 10a-5p mimic and inhibitor. (E,F) The fusion index (%) of chicken myoblasts after transfection with miRNA-10a-5p mimic and inhibitor. (G,H) The statistical results of the myotube area of myoblasts after transfection with miRNA-10a-5p mimic and inhibitor. (I,J) The image of IFA under fluorescence inverted microscope after transfection with miRNA-10a-5p mimic and inhibitor. In all graphs, the results are shown as mean ± SEM (standard error of the mean) (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies and their dilution ratios were as follows: MYHC rabbit polyclonal antibody (25182-1-AP, Proteintech, Wuhan, China, 1:500), BCL6 rabbit polyclonal antibody (bs-2734R, Bioss, Beijing, China, 1:500), GAPDH rabbit polyclonal antibody (ET1601-4, HUABIO, Hangzhou, China, 1:500), and HRP-binding Mouse anti-rabbit IgG (bsm-33179M-HRP, Bioss, Beijing, China, 1:2000).

Techniques: Quantitative RT-PCR, Marker, Transfection, Expressing, Inverted Microscopy

Journal: International journal of molecular sciences

Article Title: miRNA-10a-5p Targeting the BCL6 Gene Regulates Proliferation, Differentiation and Apoptosis of Chicken Myoblasts.

doi: 10.3390/ijms23179545

Figure Lengend Snippet: Figure 6. BCL6 gene promotes chicken myoblasts’ differentiation. (A,B) The results of qRT-PCR for differentiation marker genes MYOD1 and MYOG after transfection with pcDNA3.1-BCL6 and siRNA-1651. (C,D) The protein expression of MYHC, MYOD 1, and GAPDH after transfection with pcDNA3.1-BCL6 and siRNA-1651. (E,F) The fusion index (%) of chicken myoblasts after transfection with miRNA-10a-5p mimic and inhibitor. (G,H) The statistical results of the myotube area of myoblasts after transfection with pcDNA3.1-BCL6 and siRNA-1651. (I,J) The results of IFA after transfection with pcDNA3.1-BCL6 and siRNA-1651. In all graphs, the results are shown as mean ± SEM (standard error of the mean) (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies and their dilution ratios were as follows: MYHC rabbit polyclonal antibody (25182-1-AP, Proteintech, Wuhan, China, 1:500), BCL6 rabbit polyclonal antibody (bs-2734R, Bioss, Beijing, China, 1:500), GAPDH rabbit polyclonal antibody (ET1601-4, HUABIO, Hangzhou, China, 1:500), and HRP-binding Mouse anti-rabbit IgG (bsm-33179M-HRP, Bioss, Beijing, China, 1:2000).

Techniques: Quantitative RT-PCR, Marker, Transfection, Expressing

Journal: Cellular microbiology

Article Title: Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage.

doi: 10.1111/cmi.12342

Figure Lengend Snippet: Fig. 1. Terminal differentiation impacts T. gondii intracellular replication in SkMCs. C2C12 SkMCs were differentiated to polynucleated myotubes and together with proliferating C2C12 myoblasts and NIH/3T3 fibroblasts were infected with T. gondii at a parasite-to-host cell ratio of 5:1. A. Genomic DNA was isolated at different time points post infection (p.i.) from myotubes (closed circles), myoblasts (closed triangles) and fibroblasts (open triangles), and the 529 bp repetitive DNA element was amplified by quantitative real-time PCR. The increase of T. gondii DNA between 4 h and subsequent time points was calculated according to the 2ΔCP method. Data represent means ± S.E.M. (n = 6); significant differences in DNA increase over time were identified by Student’s t-test (**P < 0.01; ns: not significant). B. Cells were fixed at the indicated time points and parasites were immunolabelled (green fluorescence) using a polyclonal anti-T. gondii antiserum. Host cells and parasites were also visualized using propidium iodide (red fluorescence). Representative images of both labellings were recorded by confocal laser scanning microscopy and were superimposed. C. After cells had been fluorescently labelled as described (B), the average size of parasitophorous vacuoles (PVs) in each sample was determined by counting the number of parasites in 100 PVs. Data represent means ± S.E.M. from three independent experiments; significant differences over time were identified by Student’s t-test (*P < 0.05, **P < 0.01).

Article Snippet: Cells were incubated overnight at 4°C with monoclonal mouse anti-BrdU (1:1400 in 1% BSA in PBS; Cell Signaling Technology, Frankfurt/Main, Germany) and rabbit polyclonal anti-MyHC (1:100; see above).

Techniques: Infection, Isolation, Real-time Polymerase Chain Reaction, Confocal Laser Scanning Microscopy

Journal: Cellular microbiology

Article Title: Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage.

doi: 10.1111/cmi.12342

Figure Lengend Snippet: Fig. 2. Toxoplasma stage conversion and tissue cyst formation is preferentially triggered in mature myotubes. C2C12 terminally differentiated myotubes, C2C12 myoblasts and NIH/3T3 fibroblasts were infected with T. gondii for different periods of time as indicated. A and B. Total RNA was isolated from myotubes (closed circles), myoblasts (closed triangles) and fibroblasts (open triangles), mRNA was reverse transcribed, and cDNA was amplified using specific primers for T. gondii bradyzoite antigen 1 (BAG1; A), T. gondii enolase 1 (ENO1; B) and T. gondii tubulin by quantitative real-time PCR. The increase in BAG1 or ENO1 mRNA between 4 h and subsequent time points was calculated (2ΔCP method) and was normalized to tubulin mRNA. Results are depicted as means ± S.E.M. from five independent experiments; statistical differences were identified by ANOVA. Cell types marked with variable letters differ significantly. C. After fixation, tissue cysts of T. gondii were labelled with FITC-conjugated Dolichos biflorus lectin (DBL; green fluorescence), parasites were labelled with a polyclonal anti-Toxoplasma antiserum and Cy5-conjugated secondary antibodies (blue fluorescence), and mature myotubes were labelled with anti-myosin heavy chain and Cy3-conjugated secondary antibodies (red fluorescence). In some experiments, the cyst wall was labelled using the rat monoclonal antibody CC2 and Cy2-conjugated anti-rat IgG (right panel). Representative images of all labellings were recorded by confocal laser scanning microscopy and were superimposed. D. The percentages of DBL-reactive tissue cysts were calculated after counting the parasitophorous vacuoles (PVs) in 10 fields of vision of samples treated as described (C). Data represent means ± S.E.M. from three independent replicates; statistically significant differences between cell types were identified by ANOVA. Cell types marked with distinct letters differ significantly. nd: not detected.

Article Snippet: Cells were incubated overnight at 4°C with monoclonal mouse anti-BrdU (1:1400 in 1% BSA in PBS; Cell Signaling Technology, Frankfurt/Main, Germany) and rabbit polyclonal anti-MyHC (1:100; see above).

Techniques: Infection, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Confocal Laser Scanning Microscopy

Journal: Cellular microbiology

Article Title: Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage.

doi: 10.1111/cmi.12342

Figure Lengend Snippet: Fig. 6. Tspyl2 deficiency in SkMCs facilitates intracellular replication of T. gondii. Tspyl2-deficient mutants 1 and 2, negative control transfectants and wild-type C2C12 SkMCs were induced to undergo myogenic differentiation by cell–cell contact. After 144 h of differentiation, they were infected with T. gondii at a parasite-to-host cell ratio of 3:1. A. Cells were fixed at 24 and 72 h of infection, and parasites were immunolabelled (green fluorescence) using a polyclonal anti-T. gondii antiserum. Host cells and parasites were also visualized using propidium iodide (red fluorescence). Representative images of both labellings were recorded by confocal laser scanning microscopy and were superimposed. B. By using immunofluorescence microscopy as described (A), the average size of parasitophorous vacuoles (PVs) was determined in Tspyl2-deficient mutants 1 (open squares) and 2 (open diamonds), negative control transfectants (open circles) and wild-type C2C12 SkMCs (closed circles) by counting the number of parasites in 100 PVs. Data represent means ± S.E.M. from two independent experiments; significant differences between the different cell lines were identified by ANOVA. Cells marked with distinct letters differ significantly. C. Genomic DNA was isolated at 4 and 72 h after infection and the 529 bp repetitive DNA fragment was amplified by quantitative real-time PCR. The increase of T. gondii DNA between 4 h and 72 h was calculated according to 2ΔCP. Results are means ± S.E.M. (n = 3).

Article Snippet: Cells were incubated overnight at 4°C with monoclonal mouse anti-BrdU (1:1400 in 1% BSA in PBS; Cell Signaling Technology, Frankfurt/Main, Germany) and rabbit polyclonal anti-MyHC (1:100; see above).

Techniques: Negative Control, Infection, Confocal Laser Scanning Microscopy, Microscopy, Isolation, Real-time Polymerase Chain Reaction

Journal: Cellular microbiology

Article Title: Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage.

doi: 10.1111/cmi.12342

Figure Lengend Snippet: Fig. 7. Host cell Tspyl2 is required for efficient formation of T. gondii bradyzoites and tissue cysts in SkMCs. Tspyl2-deficient C2C12 mutants 1 and 2 as well as negative control-transfected and wild-type C2C12 were induced to differentiate to mature myotubes by cell–cell contact. After 144 h, they were infected with T. gondii (parasite-to-host cell ratio 3:1). A. Cells were fixed at 96 h after infection, and tissue cysts of T. gondii were labelled with FITC-conjugated Dolichos biflorus lectin (DBL; green fluorescence), parasites were labelled with a polyclonal anti-Toxoplasma antiserum and Cy5-conjugated secondary antibodies (blue fluorescence), and mature myotubes were labelled with anti-myosin heavy chain and Cy3-conjugated secondary antibodies (red fluorescence). Representative images of all labellings were recorded by confocal laser scanning microscopy. B. Cells from different time points post infection (p.i.) were analysed as described (A). The percentages of DBL-reactive tissue cysts among the parasitophorous vacuoles (PVs) from 10 fields of vision were determined; results represent means ± S.E.M. from three independent experiments. Cells marked by distinct letters differ significantly (ANOVA). nd: not detected. C. Total RNA was isolated from Tspyl2-deficient C2C12 mutants 1 (open squares) and 2 (open diamonds), negative control transfectants (open circles) and wild-type C2C12 (closed circles) at the indicated time points. After reverse transcription, cDNA was amplified by real-time PCR with primers specific for T. gondii bradyzoite antigen 1 (BAG1). The increase in BAG1 mRNA between 4 h and subsequent time points was calculated (2ΔCP method) and was normalized to T. gondii tubulin mRNA. Results are depicted as means ± S.E.M. from three independent experiments; significant differences between means were identified by ANOVA (*P < 0.05).

Article Snippet: Cells were incubated overnight at 4°C with monoclonal mouse anti-BrdU (1:1400 in 1% BSA in PBS; Cell Signaling Technology, Frankfurt/Main, Germany) and rabbit polyclonal anti-MyHC (1:100; see above).

Techniques: Negative Control, Transfection, Infection, Confocal Laser Scanning Microscopy, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: Thyroid hormone regulates muscle fiber type conversion via miR-133a1

doi: 10.1083/jcb.201406068

Figure Lengend Snippet: TEAD1 mediates miR-133a and TH action in myofiber specification. (A and B) C2C12 myotubes were cotransfected with miR-133a mimics and/or TEAD1 with or without 3′ UTR. 48 h after transfection, immunostaining was performed. Bars, 200 µm. (C) Quantitative values were determined in four random fields for each group. (D) qRT-PCR analysis of the expression of TEAD1 and MyHC isoforms in C2C12 myotubes transfected with TEAD1 in the presence of T3. (E) qRT-PCR analysis of the expression of TEAD1 and MyHC isoforms in SOL muscles of MMI-treated mice, MMI-treated mice with T3 treatment for 5 d, and MMI-treated mice with electrotransfer of TEAD1 and T3 treatment for 5 d ( n = 3). (F) Schematic representation of miR-133a–mediated TH function in muscle fiber type determination. Means ± SD (error bars) are shown. *, P < 0.05; **, P < 0.01.

Article Snippet: Myotubes were treated with 0.1% Triton X-100 in PBS for 10 min. After blocking with 2% BSA in PBS for 30 min, the expressions of MYH, MyHCs, and MyHCf in C2C12 myotubes were detected with rabbit polyclonal MyHC (1:200, sc-20641; Santa Cruz Biotechnology, Inc.), mouse monoclonal skeletal myosin-fast (1:2,000, M4276; Sigma-Aldrich), or mouse monoclonal myosin-slow (1:2,000, M8421; Sigma-Aldrich) antibodies.

Techniques: Transfection, Immunostaining, Quantitative RT-PCR, Expressing, Muscles, Electrotransfer

Journal: The Journal of Cell Biology

Article Title: Thyroid hormone regulates muscle fiber type conversion via miR-133a1

doi: 10.1083/jcb.201406068

Figure Lengend Snippet: The effect of T3, TEAD1, miR-133a on the promoter activity of MyHC-1. (A) Mouse MyHC-I gene promoter containing an MCAT element and putative TREs. (B) C2C12 myoblasts cultured in Td medium transfected with pGL3-Basic or a reporter containing TRE region. 24 h after transfection, T3 was added for 1 d. Promoter activities were evaluated with a luciferase assay. (C) C2C12 myoblasts were cotransfected with a reporter containing MyHC-I promoter and TEAD1 expression vector. Promoter activities were determined with a luciferase assay. (D) A ChIP assay was performed using chromatin from C2C12 myotubes. Anti-TEAD1, normal mouse IgG, and anti-TR (C4) antibodies were used for immunoprecipitation. Purified DNA was then analyzed by PCR using two sets of primers specific for the MCAT region. Water was used as a negative control for PCR (empty). (E) C2C12 myoblasts were cotransfected with miR-133a mimics or mimics control, and reporters containing MyHC-I promoter, MCAT element, or TRE region as indicated. Promoter activities were examined with a luciferase assay. Means ± SD (error bars) are shown. **, P < 0.01.

Article Snippet: Myotubes were treated with 0.1% Triton X-100 in PBS for 10 min. After blocking with 2% BSA in PBS for 30 min, the expressions of MYH, MyHCs, and MyHCf in C2C12 myotubes were detected with rabbit polyclonal MyHC (1:200, sc-20641; Santa Cruz Biotechnology, Inc.), mouse monoclonal skeletal myosin-fast (1:2,000, M4276; Sigma-Aldrich), or mouse monoclonal myosin-slow (1:2,000, M8421; Sigma-Aldrich) antibodies.

Techniques: Activity Assay, Cell Culture, Transfection, Luciferase, Expressing, Plasmid Preparation, Immunoprecipitation, Purification, Negative Control, Control