anti mrf4  (ABclonal Biotechnology)

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: RMS survival is selectively regulated by MyoD (A) Representative images of immunohistochemistry staining of MyoD and myogenin in patient embryonal and alveolar tumors. (B) Histologic images in (A) were scored for MyoD and myogenin immunohistochemistry staining positivity, n = 5. (C) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA targeting myogenin were treated with TNF and DOX for 24 h and subsequently measured for cell death, n = 3. (D) RNA was prepared from cells in (C) and qPCR analysis was performed probing for myogenin and its target genes, n = 3. (E) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA against either MyoD, MYF5, myogenin, MRF4, MEF2C, and MEF2D (sh-MRFs) were treated with TNF for 24 h and cell death was subsequently measured, n = 3. (F) CHRONOS Score for MRFs in sarcoma cell lines tested in DepMap. RMS cell lines are highlighted in purple, with a score of < −1 indicating a gene that is essential; ATRT, atypical teratoid rhabdoid tumors; ES, Ewing sarcoma; FS, follicular sarcoma; LMS, leiomyosarcoma; LS, liposarcoma; MRT, malignant rhabdoid tumor; PS, pleomorphic sarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma; TS, thyroid sarcoma; US, undifferentiated sarcoma. (G and H) Kaplan-Meier curve, log rank test, showing the correlation of both (G) MyoD and (H) myogenin expression stratified by median patient expression to overall survival of rhabdomyosarcoma patients from an R2 Genomics Analysis and Visualization Platform. (I) CHRONOS Score for p65 in sarcoma cell lines tested in the Cancer Dependency Map. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Article Snippet: Antibodies used in this study included, IκBα (Cell Signaling Technology Cat# 9242, RRID: AB_331623 ), α-tubulin (Thermo Fisher Scientific Cat# A11126, RRID: AB_2534135 ), p65/RelA (Santa Cruz Biotechnology Cat# sc-109, RRID: AB_632039 ), MyoD (Santa Cruz Biotechnology Cat# sc-377460, RRID: AB_2813894 ), Myf5 (Santa Cruz Biotechnology Cat# sc-518039), myogenin (Santa Cruz Biotechnology Cat# sc-52903, RRID: AB_784707 ), MRF4 (Santa Cruz Biotechnology sc-514379), MEF2C (Abcam Cat# ab211493, RRID: AB_2864417 ), MEF2D (Santa Cruz Biotechnology Cat# sc-271153, RRID: AB_10614669 ), MYHC (Sigma-Aldrich Cat# M8421, RRID: AB_477248 ), TnnT (Sigma-Aldrich Cat# SAB4200717, RRID: AB_2892079 ), CYLD (Cell Signaling Technology Cat# 4495, RRID: AB_10557111 ), p -RIPK1 (Proteintech Cat# 66854-1-Ig, RRID: AB_2882194 ), RIPK1 (Thermo Fisher Scientific Cat# PA5-20811, RRID: AB_11154790 ), DNMT1 (Novus Cat# NB100-56519, RRID: AB_838131 ), DNMT3A (Santa Cruz Biotechnology Cat# sc-365769, RRID: AB_10844010 ), DNMT3B (Cell Signaling Technology Cat# 67259, RRID: AB_2799723 ), and HRP conjugated secondary antibodies anti-mouse IgG (Promega Cat# W4021, RRID: AB_430834 ) and anti-rabbit IgG (Promega Cat# W4011, RRID: AB_430833 ).

Techniques: Immunohistochemistry, Staining, Expressing, Control

Journal: Poultry Science

Article Title: Dysfunctional terminal differentiation of satellite cells in the wooden breast muscle of Ross 308 broiler chickens

doi: 10.1016/j.psj.2025.105041

Figure Lengend Snippet: Sequences of the primers for qRT-PCR.

Article Snippet: After a brief wash in TBST, the primary antibodies against anti-mouse Pax7 monoclonal antibody (DSHB, Developmental Studies Hybridoma Bank, IA), anti-rabbit MyoG polyclonal antibody (Aifang Biological Co., Ltd., Changsha, China), anti-rabbit MRF4 polyclonal antibody (Proteintech Group Inc., Wuhan, China), anti-rabbit MEF2C polyclonal antibody (Abclonal Technology Co., Ltd., Wuhan, China), anti-rabbit GAPDH polyclonal antibody (Proteintech Group Inc.) were incubated at 4 °C overnight with gentle shaking.

Techniques: Sequencing

Journal: Poultry Science

Article Title: Dysfunctional terminal differentiation of satellite cells in the wooden breast muscle of Ross 308 broiler chickens

doi: 10.1016/j.psj.2025.105041

Figure Lengend Snippet: Gene and protein expression of regulatory factors related to differentiation and fusion of muscle satellite cells in normal (NOR) breast muscle, moderate (MOD) and severe (SEV) degree of wooden breast muscle. (A) Relative mRNA expression of MRFs family gene Myf5, MyoD, MyoG, MRF4 and MEF2C . (B, C) Representative western blotting images and relative protein expression of differentiation-related proteins. (C) Relative mRNA expression of fusion-related genes. Data are presented as mean ± SEM (n = 8), and different letters (a, b) indicate significant differences ( P < 0.05). Abbreviations: GAPDH , glyceraldehyde-3-phosphate dehydrogenase; MEF2C , myocyte specific enhancer factor 2C; MRF4 , myogenic regulatory factor 4; Myf5 , myogenic factor 5; MyoD , myogenic differentiation; MyoG , myogenin; SEM, standard error of the mean.

Article Snippet: After a brief wash in TBST, the primary antibodies against anti-mouse Pax7 monoclonal antibody (DSHB, Developmental Studies Hybridoma Bank, IA), anti-rabbit MyoG polyclonal antibody (Aifang Biological Co., Ltd., Changsha, China), anti-rabbit MRF4 polyclonal antibody (Proteintech Group Inc., Wuhan, China), anti-rabbit MEF2C polyclonal antibody (Abclonal Technology Co., Ltd., Wuhan, China), anti-rabbit GAPDH polyclonal antibody (Proteintech Group Inc.) were incubated at 4 °C overnight with gentle shaking.

Techniques: Expressing, Western Blot

Journal: Stem Cells International

Article Title: Noggin Combined With Human Dental Pulp Stem Cells to Promote Skeletal Muscle Regeneration

doi: 10.1155/sci/2812390

Figure Lengend Snippet: Noggin accelerates the progress of myogenic differentiation of DPSCs. (a–c) The levels of relative mRNAs, including (a) MyoD, (b) Desmin, and (c) MRF4, were assessed using quantitative PCR ( n = 3). (d, e) Immunofluorescence staining with MyoD (red) (d) or Desmin (red) (e) and DAPI (blue) on Day 14 comparing 5-Aza induction with or without Noggin (200 ng/mL), respectively. Arrowheads indicate merged MyoD-positive myonuclei; white dotted line indicates cell outline; green dotted circle indicates Desmin-positive merged myonuclei; white scale bar, 50 µm; green scale bar, 10 µm. (f, g) Percentage of MyoD-positive cells (f) or Desmin-positive myotube-like cells (g) is presented as the ratio of nuclei to total nuclei ( n = 4). (h) Protein expression of Desmin and MRF4 was assessed using western blot on Days 14 and 21. (i, j) Semiquantitative analysis of the expression ratio of (i) Desmin/ β -actin and (j) MRF4/ β -actin ( n = 4). The differences between blank and 5-Aza or between 5-Aza, 5-Aza + 100N and 5-Aza + 200N were presented as ⁣ ∗ p < 0.05 and ⁣ ∗∗ p < 0.01. 100N = 100 ng/mL Noggin; 200N = 200 ng/mL Noggin. DPSCs, dental pulp stem cells; MyoD, myogenic differentiation 1.

Article Snippet: Antibodies against β -actin (abs830031; Absin Bioscience, Shanghai, China), Pax7 (ab187339), and Myf5 (ab125078) from Abcam; MyHC IIB (BF-F3) and MyHC IIA (SC-71) from Developmental Studies Hybridoma Bank (DSHB, ID, USA); p-Smad 1/5/9 (#13820) and Smad 1 (#6944) from Cell Signaling Technology (CST, MA, USA); and MyoD (#18943), Desmin (#60226), MRF4 (#11754), Pax3 (#21383), Six1 (#10709), Eya2 (#11314), ID1 (#18475), and MSX1 (#17678) from Proteintech (IL, USA) were used.

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

doi: 10.1371/journal.pone.0081713

Figure Lengend Snippet: (A) Detection of full length MSV mRNA in a stable MSV over-expressing (MSV-line) and an empty vector stably transfected C 2 C 12 myoblast line (Control-line) using RT-PCR. GAPDH was used as a positive control for each sample. NTC is a no template PCR control. (B) Effect of endogenous over-expression of MSV on the proliferation of C 2 C 12 myoblasts. Proliferation of the MSV- and Control-line was determined at 0, 24, 48 and 72 h using the WST-1 cell proliferation reagent (**P<0.01, ***P<0.001, n = 8). (C) Effect of rMSV on the proliferation of C 2 C 12 myoblasts. C 2 C 12 myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (*P<0.05, **P<0.01, ***P<0.001, n = 8). (D) Effect of rMSV on the proliferation of sheep myoblasts. Sheep myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (***P<0.001, n = 8). (E) The abundance of CDK2 protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (F) The abundance of Cyclin E protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, **P<0.001, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (G) The abundance of Myf5, MyoD, Myogenin, MRF4, Pax7 and MEF2 proteins was determined using Western immunoblotting in proliferating myoblasts of the MSV- and Control-line (*P<0.05, n = 3).

Article Snippet: After washing in Tris buffered saline with 0.1% Tween 20 (TBST), the blots were blocked in blocking buffer (TBST buffer supplemented with 1% PVP-10, 1% PEG4000, 0.3% BSA fraction V, 0.01% Thimerasol) or with TBST buffer supplemented with 5% non-fat milk powder for one hour, and then incubated with different dilutions of primary antibodies: anti-MSVab, anti-myostatin , anti-CDK2 (sc-163, Santa Cruz Biotechnology), anti-Cyclin E (sc-481, Santa Cruz Biotechnology), anti-Myf5 (sc-302, Santa Cruz Biotechnology), anti-MyoD (sc-304, Santa Cruz Biotechnology), anti-MRF4 (sc-784, Santa Cruz Biotechnology), anti-MEF2 (sc-313, Santa Cruz Biotechnology), anti-Pax7 (sc-163, Santa Cruz Biotechnology), anti-Smad2/3 (sc-6032, Santa Cruz Biotechnology), anti-pSmad2/3 (Ser423/425, sc-11769R, Santa Cruz Biotechnology), anti-V5 (Invitrogen), anti-actin (A2066, Sigma), anti-SP-1 (sc-59, Santa Cruz Biotechnology) or anti-α-tubulin (T9026, Sigma) primary antibodies in the appropriate blocking buffer at 4°C overnight.

Techniques: Expressing, Plasmid Preparation, Stable Transfection, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Over Expression, Western Blot