Figure 4. Persistent PKA activation for... | Rockefeller University Press

Figure 4.

$Persistent PKA activation for 15 h induced microtubule reorganization in ventricular myocytes. (A) Left: Immunoblot images of cytosolic fraction from CON and PKA myocytes in two independent experiments. Antibodies targeted EB1, total α-tubulin (α-Tub), and detyrosinated α-tubulin (deY α-Tub). CB confirms even loading. In the immunoblot image of α-Tub, and immunoblot images in the following figures, the dotted vertical line indicates lane(s) in between removed for presentation (corresponding uncropped images are shown in source data). Right: PKA:CON ratios of band intensities pooled from the number of immunoblots shown in parentheses. (B) XY and YZ plane images of CON and PKA myocytes immunostained for EB1, β-tubulin (β-Tub), and deY α-Tub. “Tubulin merge” is combined β-Tub and deY α-Tub signals. (C) Reduction of interfibrillar microtubules in PKA versus CON myocytes. Top: XY and YZ plane images of β-Tub immunofluorescence in CON and PKA myocytes. In the PKA myocyte’s YZ-plane view, the β-Tub dense region was part of an intercalated disc (ICD, based on nCadherin immunostaining, not shown). Bottom: Microtubule density quantified as “% myocyte central area occupied by β-Tub puncta,” where “myocyte central area” was defined as the cross-sectional area within 1 μm from cell contour. Numbers in parentheses are those of the myocytes studied. (D) Immunofluorescence signals of β-Tub, deY α-Tub, and their merge at a z plane close to the surface of CON and PKA myocytes. Listed P values are from t tests against null hypothesis (A), or PKA versus CON (C). Scale bars are 10 μm for B, 2 μm for C, and 5 μm for D. Source data are available for this figure: SourceData F4.$

Persistent PKA activation for 15 h induced microtubule reorganization in ventricular myocytes. (A) Left: Immunoblot images of cytosolic fraction from CON and PKA myocytes in two independent experiments. Antibodies targeted EB1, total α-tubulin (α-Tub), and detyrosinated α-tubulin (deY α-Tub). CB confirms even loading. In the immunoblot image of α-Tub, and immunoblot images in the following figures, the dotted vertical line indicates lane(s) in between removed for presentation (corresponding uncropped images are shown in source data). Right: PKA:CON ratios of band intensities pooled from the number of immunoblots shown in parentheses. (B) XY and YZ plane images of CON and PKA myocytes immunostained for EB1, β-tubulin (β-Tub), and deY α-Tub. “Tubulin merge” is combined β-Tub and deY α-Tub signals. (C) Reduction of interfibrillar microtubules in PKA versus CON myocytes. Top: XY and YZ plane images of β-Tub immunofluorescence in CON and PKA myocytes. In the PKA myocyte’s YZ-plane view, the β-Tub dense region was part of an intercalated disc (ICD, based on nCadherin immunostaining, not shown). Bottom: Microtubule density quantified as “% myocyte central area occupied by β-Tub puncta,” where “myocyte central area” was defined as the cross-sectional area within 1 μm from cell contour. Numbers in parentheses are those of the myocytes studied. (D) Immunofluorescence signals of β-Tub, deY α-Tub, and their merge at a z plane close to the surface of CON and PKA myocytes. Listed P values are from t tests against null hypothesis (A), or PKA versus CON (C). Scale bars are 10 μm for B, 2 μm for C, and 5 μm for D. Source data are available for this figure: SourceData F4.