Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle fibrosis and hypertrophy. ( A ) Picro-Sirius Red staining. Scale bar = 200 µm. ( B ) Quantification of Picro-Sirius Red staining. mRNA levels of ( C ) αSMA and ( D ) TGF-β1, two markers of fibrosis. ELISA assays were used to quantify ( E ) TGF-β and ( F ) the active phosphorylated form of SMAD2 (P¬SMAD2), a transcription factor mainly involved in TGF-β signalling. mRNA levels of ( G ) BNP and ( H ) ANP, two markers of hypertrophy. ( I ) ELISA assay was also used to quantify the levels of ANP. The percentage of stained areas was calculated in cardiac muscle sections, and the subsequent ratios are presented as relative expressions to WT values. mRNA levels were normalised to cyclophilin, and the subsequent ratios were presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on ApN receptors and signalling in the dystrophic cardiac muscle. mRNA levels of ( A ) AdipoR1 and ( B ) AdipoR2, adiponectin main receptors. ELISA assays were used to quantify ( C ) AdipoR1 and ( D ) AdipoR2. ( E ) The ratio of AdpoR1 over AdipoR2 mRNA levels was calculated within the cardiac muscle. ELISA assays were used to quantify ( F ) the active phosphorylated form of AMPKα (P-AMPK), ( G ) calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and ( H ) peroxisome proliferator-activated receptor alpha (PPARα), ApN/AdipoRon, main signalling pathways in muscle. ( I ) mRNA levels of PGC-1α. ELISA assays were used to quantify ( J ) PGC-1α, ( K ) the active phosphorylated form of the p65 subunit of NF-κB (P-p65), a transcription factor mainly involved in inflammation, and ( L ) utrophin A (UTRN), a dystrophin analogue. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expression to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle oxidative capacity, injury, and overall muscle function. mRNA levels of ( A ) ERRα and ( B ) mtTFA, two markers of mitochondrial biogenesis. ELISA assay was used to quantify ( C ) TOMM20, a marker of mitochondrial content. ( D ) Wire test where mice hanging time was recorded (s). ( E ) Fore-limb grip test and ( F ) fore- and hind-limb grip test, measuring muscle strength expressed in Gram-force relative to body weight (gf/gBW). ( G ) Treadmill running exercise, where the total distance covered on the third day was measured (m). ( H ) CK and ( I ) LDH plasma activities assessing muscle injury and expressed as IU/L. ( J ) ELISA assay was used to quantify the active phosphorylated form of RIP (P-RIP), an important regulator of cellular stress that triggers a regulated pathway for necrotic cell death called necroptosis. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all ex vivo experiments. Data are means ± SD; n = 7–8 mice per group for all in vivo functional tests. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Ex Vivo, In Vivo, Functional Assay

Journal: The Journal of Cell Biology

Article Title: v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

doi: 10.1083/jcb.200102027

Figure Lengend Snippet: Analysis of the phosphorylation of GST–Cx43CT by activated Src in vitro. (A) Amino acid sequence of two predicted tryptic peptides of Cx43CT (S 244 -K 258 and Y 265 -K 287 ) that contain the putative pTyr sites (Y247 and Y265) in the phosphorylated GST–Cx43CT fusion protein. Cx43CT fused to GST begins at V236 in the cytoplasmic tail of Cx43. The amino acids deleted in the Cx43CT deletion mutant are bracketed (P 253 LSP 256 ). (B) Two-dimensional phosphotryptic peptide analysis of GST fusion proteins with wt or the Cx43CT deletion mutation phosphorylated by activated Src. Phosphotryptic peptides were resolved by electrophoresis (dimension 1) followed by ascending chromatography (dimension 2). Major phosphotryptic peptides are numbered. (C) Phosphoamino acid content of wt or site-directed mutant GST–Cx43CT phosphorylated by activated Src. Equal amounts of the GST–Cx43CT substrates were phosphorylated by Src, acid-hydrolyzed, and separated by electrophoresis at pH 1.9 (dimension 1) followed by electrophoresis at pH 3.5 (dimension 2). The migration positions of the unlabeled pSer, pThr, and pTyr standards are indicated. (D) Phosphotryptic peptide maps of wt and mutant GST–Cx43CT phosphorylated by activated Src. Resolved peptides were from one type of GST–Cx43CT or a mixture of wt and the Y265F GST–Cx43CT mutant. Peptides are numbered as in B.

Article Snippet: Activated MAP kinase was detected with a rabbit antibody specific for the phosphorylated activated form of MAP kinase (Promega) using the ECL-plus immunoblotting kit and Eastman Kodak Co. X-Omat XAR-5 films.

Techniques: Phospho-proteomics, In Vitro, Sequencing, Mutagenesis, Electrophoresis, Chromatography, Migration

Journal: The Journal of Cell Biology

Article Title: v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication

doi: 10.1083/jcb.200102027

Figure Lengend Snippet: The role of MAP kinase in the disruption of GJC by v-Src. Cells were untreated or treated with EGF or the MEK inhibitor before being lysed. The same amount of whole cell lysate was loaded for each sample. Activated MAP kinase was detected with an antibody recognizing phosphorylated MAP kinase. Lane 1, untreated wtC1; lane 2, wtC1 treated with 100 ng/ml EGF for 2 min; lane 3, untreated wtS2; and lanes 4 and 5, duplicate plates of wtS2 treated with 100 μM MEK inhibitor for 1 h. The same membrane used to detect active MAP kinase was stripped and reprobed with an antibody recognizing all isoforms of p42 MAP kinase. The ratios of active MAP kinase to total MAP kinase were determined and normalized to the untreated wtS2 cells set as 100%. Average GJC values obtained from multiple plates are reported for lanes 1, 3, and 4, and the GJC value from a single plate that was also used to measure the level of activated MAP kinase is shown for lane 5. The number of injections contributing to the determination of GJC is shown in brackets. Active MAP kinase was reduced to 5 and 2% of the starting level by the 1 h treatment with MEK inhibitor as shown in lanes 4 and 5, respectively.

Article Snippet: Activated MAP kinase was detected with a rabbit antibody specific for the phosphorylated activated form of MAP kinase (Promega) using the ECL-plus immunoblotting kit and Eastman Kodak Co. X-Omat XAR-5 films.

Techniques: Disruption, Membrane