monoclonal antibodies against erb  (Bio-Rad)

Journal: bioRxiv

Article Title: Rev-erb-α controls skeletal muscle calcium homeostasis through myoregulin repression: implications in Duchenne Muscular Dystrophy

doi: 10.1101/2021.04.22.440922

Figure Lengend Snippet: ( A ) RyR1, ( B ) Serca1 and ( C ) Serca2 gene expression in muscle from Rev-erbα +/+ and Rev-erbα -/- mice (n=6, NS by unpaired t-test), ( D ) RyR1, ( E ) Serca1 and ( F ) Serca2 gene expression levels in pBabe and REV-ERBα overexpressing differentiated C2C12 (n=3, NS by unpaired t-test). Mln expression in ( G ) muscle from Rev-erbα +/+ and Rev-erbα -/- mice (n=6, **p=0.0025 compared to Rev-erbα +/+ , unpaired t-test), ( H ) muscle from SR9009 treated wild-type (WT) animals (n=5, **p=0.0069 compared to vehicle, unpaired t-test), ( I ) REV-ERBα overexpressing C2C12 (n=5, **p=0.0023 vs. pBabe, unpaired t-test), ( J-K ) C2C12 treated either with ( J ) 10μM of the Rev-erb agonist SR9009 (n=3, *p=0.0155) or ( K ) 10μM of the Rev-erb antagonist SR8278 (n=6, ***p<0.0001 vs. DMSO-treated cells, unpaired t-test). ( L ) Schematic representation of the Mln promoter indicating the presence of three putative Rev-erb-α Response Elements (RevRE), located ~1.4, ~5.4kb and ~6.7kb upstream the transcription initiation site. ( M ) Chromatin ImmunoPrecipitation analysis using an anti-Rev-erb-α antibody or control Immunoglobulin G (IgG) and specific primers targeting the three identified putative sites or a negative control region located ~9kb upstream the transcription initiation site. n=6-8 mice, data are means ± SEM, site −6.7kb **p=0.0017, site −5.4kb ***p<0.0001, site −1.4kb ***p=0.001 vs. IgG. ( N ) Mln expression in mice with muscle-specific expression of a mutated isoform of Rev-erb-α lacking the DNA binding domain ( Rev-erbα DBD mut fl/fl , MCK Cre/+ ) and control Rev-erbα DBD mut fl/fl mice. n=3-5, *p=0.0139 vs. Rev-erbα DBD mut fl/fl , unpaired t-test. ( O ) Thapsigargin (TG)-induced Sarcoplasmic Reticulum (SR) Ca 2+ release in pBabe and Mln overexpressing differentiated C2C12. Cells are loaded with Fluo4-AM and SR Ca 2+ release is induced by the addition of 1μM TG. Results are expressed as means ± SEM of the Delta F/F0 ratio, n=7. ( P ) Peak fluorescence intensity of thapsigargin (TG)-induced Sarcoplasmic Reticulum (SR) Ca 2+ release in pBabe Mln overexpressing differentiated C2C12, normalized to pBabe and expressed as mean ± SEM. n=7, **p=0.024 vs. pBabe, unpaired t-test. ( Q ) Thapsigargin (TG)-induced Sarcoplasmic Reticulum (SR) Ca 2+ release in pBabe, REV-ERBα overexpressing and REV-ERBα/Mln overexpressing differentiated C2C12. Cells are loaded with Fluo4-AM and SR Ca 2+ release is induced by the addition of 1μM TG. Results are expressed as means ± SEM of the Delta F/F0 ratio of 6 independent experiments. ( R ) Peak fluorescence intensity of thapsigargin (TG)-induced Sarcoplasmic Reticulum (SR) Ca 2+ release in pBabe, REV-ERBα overexpressing and REV-ERBα/Mln overexpressing differentiated C2C12, normalized to pBabe and displayed as means ± SEM. n=6, *p<0.026 vs. pBabe, $p<0.0293 vs. REV-ERBα, 1-way ANOVA, Tukey’s multiple comparison test.

Article Snippet: 50μg of chromatin were immunoprecipitated overnight at 4°C with an antibody against Rev-erb-α (13418S, Cell signaling Technology, Danvers, Massachusetts, United States).

Techniques: Gene Expression, Expressing, Chromatin Immunoprecipitation, Control, Negative Control, Binding Assay, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Rev-erb-α controls skeletal muscle calcium homeostasis through myoregulin repression: implications in Duchenne Muscular Dystrophy

doi: 10.1101/2021.04.22.440922

Figure Lengend Snippet: ( A ) NR1D1 ( REV-ERB-α ) expression in muscle biopsies from controls (n=14) and patients suffering from Duchenne Muscular Dystrophy (DMD, n=23), **p=0.0092, unpaired t-test, data from GEO DataSets GSE6011. ( B ) NR1D1 expression in control or DMD myoblasts, n=5-7. *p=0.0404 vs. control cells in panel B, unpaired t-test. ( C ) Representative curves and ( D ) peak fluorescence intensity of thapsigargin (TG)-induced Sarcoplasmic Reticulum (SR) Ca 2+ release in myoblasts from controls or patients suffering from Duchenne Muscular Dystrophy (DMD) treated with SR9009 (10μM) or vehicle. Cells are loaded with Fluo4-AM and SR Ca 2+ release is induced by the addition of 1μM TG. Results are expressed as means ± SEM of the Delta F/F0 ratio, n=3 controls, n=7 in both DMD groups. **p=0.0049 vs. control cells, unpaired t-test, $p=0.0138 vs. DMD DMSO, paired t-test. ( E ) Hematoxylin and eosin and Sirius red staining of tibialis anterior muscles obtained from vehicle- and SR9009-injected mdx/Utr +/- mice. Scale bars represent 100μm. ( F ) Myofiber cross-sectional area distribution (n=7-9), *p<0.05 vs. vehicle-treated mdx/Utr +/- mice. ( G ) Circulating Creatine PhosphoKinase (CPK) activity, n=8-9, *p=0.0329 vs. vehicle-treated mdx/Utr +/- animals. ( H ) Muscular hydroxyproline, n=8-10, *p=0.0172. (I) Col1a2, ( J ) Pdgfra and ( K ) Mln gene expression; n=8-12, *p=0.0314, *p=0.0164 and *p=0.0402, respectively. ( L ) SERCA activity (n=8-10) in muscular microsomes from mdx/Utr +/- mice treated for 20 days with SR9009 (100mg/kg) or vehicle; *p=0.0266. ( M ) In situ measurement of gastrocnemius -developed force (n=4-9, *p=0.0301).

Article Snippet: 50μg of chromatin were immunoprecipitated overnight at 4°C with an antibody against Rev-erb-α (13418S, Cell signaling Technology, Danvers, Massachusetts, United States).

Techniques: Expressing, Control, Fluorescence, Staining, Muscles, Injection, Activity Assay, Gene Expression, In Situ