Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 6. DAPA alleviated ion channel disorders and atrial fibrosis caused by Ang II. A. Immunoblotting analysis of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40, smad2/3, p-smad2/3, TGF-β1 in atrial tissue. B, C. Quantification of CAMKII, ox-CAMKII expression level in atrial tissue. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. D-I. Quantification of atrial electrical activity associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J, K. Quantification of atrial fibrosis associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal-Wallis post hoc multicomparison test (B–K). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Western Blot, Expressing, Activity Assay

Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 7. SGLT2i alleviated ion channel disorders and atrial fibrosis caused by activation of CAMKII. A-I Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40 in HL-1 atrial myocytes. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J-N. Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, smad2/3, p-smad2/3, TGF-β1 in primary mouse fibroblast. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal- Wallis post hoc multicomparison test (B–I and K–N). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Activation Assay, Western Blot, Quantitation Assay, Expressing

Journal: Frontiers in Physiology

Article Title: Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

doi: 10.3389/fphys.2018.01383

Figure Lengend Snippet: Atrial profibrotic and connexin remodeling. (A) Representative Western blot examples (top) and quantification of protein expression (bottom; mean ± SEM) of periostin, vimentin, α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP9), transforming growth factor-β1 (TGF-β1), fibronectin and collagen 1α (col1a) in right-atrial tissue homogenates from Ctl (white bars), HFrEF (blue bars) or HFrEF-cAF (red/blue-striped bars) patients. Vertical white lines separate non-adjacent lanes on the same blot. (B) Representative Western blot examples (top) and quantification of protein expression (bottom; mean ± SEM) of total connexin-40 (Cx40), total and Ser368-phosphorylated connexin-43 (Cx43). GAPDH was used as loading control and is shown for the samples used for Western blots of periostin, vimentin, α-SMA and MMP9, for TGF-β1, and for fibronectin and col1a. Numbers in bars indicate number of patients. ∗ indicates P < 0.05 vs. Ctl, # indicates P < 0.05 vs. HFrEF.

Article Snippet: Protein levels of α smooth-muscle actin (αSMA, 1:500, A5228, Sigma-Aldrich, St. Louis, MO, United States), calsequestrin (CSQ, 1:2,500, PA1-913, Thermo Fisher Scientific), collagen 1α (Col1a, 1:1,000, sc-293182, Santa Cruz Biotechnology, Santa Cruz, CA, United States), connexin-40 (Cx40, 1:1,000, ab1726, Merck Millipore, Burlington, MA, United States), total and Ser368-phosphorylated connexin-43 (Cx43, 1:1,000, 3511, Cell Signaling Technology, Danvers, MA, United States), fibronectin (1:1,000, sc-8422, Santa Cruz Biotechnology), GAPDH (1:20,000, 5G4 6C5, HyTest, Turku, Finland), junctin (1:2,000, LS-C196703, LifeSpan BioSciences, Seattle, WA, United States), junctophilin-2 (1:1,000, sc-134875, Santa Cruz), matrix metallopeptidase 9 (MMP9, 1:200, ab38898, Abcam, Cambridge, United Kingdom), Na + -Ca 2+ -exchanger type-1 (NCX1, 1:1,000, R3F1, Swant, Marly, Switzerland), periostin (1:1,000, sc-134875, Santa Cruz Biotechnology), total, Ser16- and Thr17-phosphorylated phospholamban (PLB, all 1:1,000, ab2865 and ab92697, Abcam, Cambridge, United Kingdom and A010-13, Badrilla Ltd., Leeds, United Kingdom), total ryanodine receptor type-2 channel (RyR2, 1:1,000, MA3-916, Thermo Fisher Scientific), sarcolipin (1:100, ABT13, Merck Millipore), sarcoplasmic reticulum (SR) Ca 2+ -ATPase type-2a (SERCA2a; 1:2,000, sc-8095, Santa Cruz Biotechnology), transforming growth factor β1 (TGF-β1, 1:1000, ab9758, Abcam), and vimentin (1:1,000, sc373717, Santa Cruz Biotechnology) were determined using appropriate primary antibodies.

Techniques: Western Blot, Expressing

Journal: The FASEB Journal

Article Title: Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure

doi: 10.1096/fj.201700699R

Figure Lengend Snippet: Cx43 protein is increased in CCM3KD brain endothelial cells. A) Control or CCM3KD (siRNA-mediated 85% CCM3 knockdown; Supplemental Fig. 1) whole-cell lysates were prepared and analyzed via Western blot. Cx43 protein, but not Cx37 or Cx40 protein, is significantly elevated relative to control (n = 3 independent experiments). *P < 0.05 compared to control. B) RNA extracted from control and CCM3KD cells and subjected to reverse-transcription quantitative PCR revealed no significant changes in Cx43 transcript. Data represent means ± sem fold-change normalized to β-actin (Cx43: n = 6, Cx40, Cx37: n = 3 independent experiments). **P < 0.01 compared with control. C) Whole-cell lysates were subjected to cell fractionation. Cx43 was only present in the insoluble fraction (caveolin-1) and not in the cytosol (Hsp70; representative Western blot, n = 3 independent experiments). D) FRAP analysis of control and CCM3KD cells expressing Cx43-AcGFP. FRAP was performed on membrane ROIs (n = 6 ROIs from 6 cells). E) Immunofluorescence staining of Cx43 (column 1), merged Cx43, and DAPI staining (column 2), and zoom images of Cx43 GJs along the cell membrane (column 3, dashed lines) in control and CCM3KD cells (GJ plaques are indicated with white arrows). Scale bar, 100 μm. F) Cx43 GJ plaque size was measured in ImageJ. For 3 image fields with ∼8 cells/field, the entire membrane region of every cell was selected to analyze particle (GJ plaque) size in Cx43-labeled samples. Average plaque size is reported as mean ± sem of all GJ plaques measured (∼50 plaques/field). This analysis revealed significantly larger GJ plaques in CCM3KD cells compared with controls. **P < 0.01.

Article Snippet: Primary Abs used were as follows: Cx37 (Abcam, Cambridge, MA, USA); Cx40 (GeneTex, Irvine, CA, USA); Cx43 and β-tubulin; CCM1 (Boster Bio, Pleasanton, CA, USA); CCM2 (Sigma-Aldrich); CCM3 (ProteinTech, Rosemont, IL, USA); and ZO-1 and Claudin-5 (Thermo Fisher Scientific).

Techniques: Control, Knockdown, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Fractionation, Expressing, Membrane, Immunofluorescence, Staining, Labeling