Anti-MyoD1 antibody

• WB

Ap

Western blot - Anti-MyoD1 antibody (AB64159)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab64159 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

ab64159 detects a band at 45 kDa, while this differs to its predicted molecular weight of 34kDa, the banding pattern observed is consistent with what has been described in the literature PMID : 19352326.

All lanes:

Western blot - Anti-MyoD1 antibody (ab64159) at 1 µg/mL

Lane 1:

Skeletal Muscle (Mouse) Tissue Lysate at 20 µg

Lane 2:

Rh30 Whole Cell Lysate at 5 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 34 kDa

Observed band size: 45 kDa,47 kDa

true

Exposure time: 1min

• sELISA

Unknown

Sandwich ELISA - Anti-MyoD1 antibody (AB64159)

Standard Curve for Myo-D; dilution range 1 pg/ml to 1 ug/ml using Capture Antibody Mouse monoclonal [5.2F] to MyoD1 (ab16148) at 5ug/ml and Detector Antibody Rabbit polyclonal to MyoD1 (ab64159) at 0.1ug/ml.

• WB

CiteAb

Western blot - Anti-MyoD1 antibody (AB64159)

Western Blotting using Anti-MyoD1 antibody, ab64159. Publication image from Zhang, H. et al., 2020, Cell Res, 32839552. Legend direct from paper.

Necroptosis-deficient mice exhibit muscle regeneration defects.a Representative of H&E staining of TA muscle cross-sections from both uninjured (Day 0) and injured (7 and 15 days after CTX injection) WT and Mlkl−/− mice. Scale bars, 50 µm. b Quantification of myofiber sizes from cross-sectional areas (CSAs) of injured mice (7 days after CTX injection) and myofibers with multiple central nuclei out of total cells with central nuclei (in vivo fusion index) 15 days post CTX injection. 3 different views were counted for each mouse. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. c Immunofluorescence staining of MYH3 (green) and Laminin (red) in TA muscle cross sections from both uninjured (Day 0) and injured (4 days after CTX injection) mice. Nuclei were identified by staining with DAPI. Scale bars, 25 µm. d Quantification of the MYH3+ myofiber sizes from CSAs of injured mice (4 days after CTX injection, as representatively shown in c). The sizes of 200 adjacent MYH3+ myofibers were measured for each mouse. Each dot represents an individual myofiber. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. e Immunoblotting analysis of MyoD, MyoG, and MYH3 expression in uninjured (Day 0) or injured (4 days after CTX injection) TA muscles using antibodies as indicated. Whole TA muscle lysates were exacted, as described in Materials and Methods, from 3 mice and pooled together for each condition. HSP70 serves as the loading control. The asterisk (*) denotes the non-specific band. Experiments were repeated independently for three times. f Quantification of the MuSCs in injured TA muscles (3 days after CTX injection) by FACS analysis. MuSCs were isolated as described in Materials and Methods. The histogram represents the percentage of MuSCs (PI−CD11b−CD31−CD45−Sca1−Vcam+ population) out of the total digested mono-nucleus cells. Each dot represents an individual mouse. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. g Immunofluorescence staining of Ki67 (green) and Pax7 (red) in freshly isolated MuSCs (3 days after CTX injection). MuSCs were isolated by FACS and fixed on PDL/Collagen-I pre-coated coverslip, as described in Materials and Methods, followed by immunofluorescence staining using the antibodies as indicated. Nuclei were identified by staining with DAPI. Scale bars, 5 µm. h Quantification of the Ki67+/Pax7+ cells as shown in g. MuSCs isolated from 3 mice were pooled together for immunofluorescence staining in each group. The histogram represents the percentage of Ki67+/Pax7+ cells out of 200 Pax7+cells per genotype. The data are expressed as the means ± SD of 6 images. i qRT-PCR analysis of Ccnd1 mRNA level in MuSCs isolated from injured mice (3 days after CTX injection). The mRNA level of Gapdh was used as the internal control. MuSCs isolated from 3 mice were pooled together for qRT-PCR analysis. The data are expressed as the means ± SD of 3 technical repeats. P values for b, d, and f were determined by unpaired two-tailed t-test; P values for h and i were determined by unpaired two-tailed t-test with Welch’s correction. **P < 0.01; ***P < 0.005. CTX, cardiotoxin; TA, tibialis anterior.

false