gsk 3β 3d10  (Cell Signaling Technology Inc)

Journal: Acta physiologica (Oxford, England)

Article Title: Cholinergic signaling impairs cardiomyocyte cohesion.

doi: 10.1111/apha.13881

Figure Lengend Snippet: FIGURE 7 CCH impaired DP-DSG2 reduction at cell borders via PI3K-AKT-GSK-3β signaling. (A) Dispase-based dissociation assay in HL-1 cardiomyocytes treated with control, CCH, Wortmannin (WM) and CCH + WM for 1 h. HL-1 cells were incubated 30 min prior to the addition of CCH with WM. Representative pictures of the wells displayed at the bottom of the bar graph, *p ≤ 0.05, one-way ANOVA with Holm-Šídák's multiple comparisons test, N = 9–10. (B) Representative Western blot showing protein expression of phospho-serine 473 of AKT (pAKTS473) and total AKT, phosphor-serine 9 of GSK-3β (pGSK-3βS9), and total GSK-3β in HL-1 cells treated as in a with respective mediators. N = 6. (C) Quantification of Western blot from b *p ≤ 0.05, one-way ANOVA with Holm-Šídák's multiple comparisons test. (D). Immunostaining in HL-1 cardiomyocytes for DP and DSG2 showing a decrease in the colocalisation of DP-DSG2 at the cell–cell contacts after 1 h CCH treatment in the presence or absence of WM. White arrows indicate DP-DSG2 colocalization at the cell membrane. Scale bar: 10 μm. (E) Quantification of DP-DSG2 at the cell–cell contacts after 1 h CCH treatment in the presence or absence of WM in HL-1 cardiomyocytes, **p ≤ 0.05, one-way ANOVA with Holm-Šídák's multiple comparisons test. Each data point represents data analyzed from one confocal image from N = 3–4 individual experiments.

Article Snippet: Following primary antibodies were added overnight at 4°C on a rocking platform: anti- AKT (Cell signaling #9272), anti- phospho- AKT (serine 473, Cell signaling #4060), anti- DP (Progen, #61003), anti- phospho- ERK1/2 ([E- 4] Santa Cruz, #sc7383), anti- ERK (Cell Signaling, #9102), anti- GSK- 3β (3D10) (Cell signaling #9832), anti- Phospho- GSK- 3β (serine 9, Cell signaling #5558), anti- phospho- PG (serine 665, self- made as described before15), anti- PG ([PG5.1] Progen #61005), anti- PKP2 (Progen, #651167), and anti- α- tubulin ([DM1A], abcam, #ab7291).

Techniques: Control, Incubation, Western Blot, Expressing, Immunostaining, Membrane

Journal: Acta physiologica (Oxford, England)

Article Title: Cholinergic signaling impairs cardiomyocyte cohesion.

doi: 10.1111/apha.13881

Figure Lengend Snippet: FIGURE 8 Inhibition of PI3K-AKT-GSK-3β signaling ameliorated CCH-mediated reduction of DSG2 binding frequency in HL-1 cells. (A) Topography images obtained during AFM measurements on HL-1 cardiomyocytes at cell borders with DSG2 coated tips after preincubating with Wortmannin for 30 min and CCH for 1 h. Green dots in topography images indicate binding events; red lines indicate cell borders. Topography images are 1.5 μm × 5 μm. (B) Quantification of DSG2 binding frequency expressed in percentage. Each data point represents 1500 curves analyzed across two areas (1.5 μm × 5 μm). *p ≤ 0.05, Student t-test with Welch's correction was performed. N = 4.

Article Snippet: Following primary antibodies were added overnight at 4°C on a rocking platform: anti- AKT (Cell signaling #9272), anti- phospho- AKT (serine 473, Cell signaling #4060), anti- DP (Progen, #61003), anti- phospho- ERK1/2 ([E- 4] Santa Cruz, #sc7383), anti- ERK (Cell Signaling, #9102), anti- GSK- 3β (3D10) (Cell signaling #9832), anti- Phospho- GSK- 3β (serine 9, Cell signaling #5558), anti- phospho- PG (serine 665, self- made as described before15), anti- PG ([PG5.1] Progen #61005), anti- PKP2 (Progen, #651167), and anti- α- tubulin ([DM1A], abcam, #ab7291).

Techniques: Inhibition, Binding Assay

Journal: Acta physiologica (Oxford, England)

Article Title: Cholinergic signaling impairs cardiomyocyte cohesion.

doi: 10.1111/apha.13881

Figure Lengend Snippet: FIGURE 9 Cholinergic signaling impairs cardiomyocyte cohesion. Cholinergic signaling impairs cardiomyocyte cohesion by (1) reducing adrenergic signaling-mediated positive adhesiotropy via inhibiting ERK1/2 activation and thereby arresting the moment of DSG2 into the cell membrane and (2) activating PI3K-AKT-mediated phosphorylation of GSK-3β at serine 9 that leads to the inhibition of DP phosphorylation by GSK-3β, which in turn results in the disruption of DP-DSG2 interaction and thereby less DSG2 at the cell borders. However, in the presence of Wortmannin, cholinergic signaling could not impair cardiomyocyte cohesion.

Article Snippet: Following primary antibodies were added overnight at 4°C on a rocking platform: anti- AKT (Cell signaling #9272), anti- phospho- AKT (serine 473, Cell signaling #4060), anti- DP (Progen, #61003), anti- phospho- ERK1/2 ([E- 4] Santa Cruz, #sc7383), anti- ERK (Cell Signaling, #9102), anti- GSK- 3β (3D10) (Cell signaling #9832), anti- Phospho- GSK- 3β (serine 9, Cell signaling #5558), anti- phospho- PG (serine 665, self- made as described before15), anti- PG ([PG5.1] Progen #61005), anti- PKP2 (Progen, #651167), and anti- α- tubulin ([DM1A], abcam, #ab7291).

Techniques: Activation Assay, Membrane, Phospho-proteomics, Inhibition, Disruption