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Results: 5

1.
Fig. 1.

Fig. 1. From: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ?2-adrenergic regulation.

Cav1.2 is enriched in caveolar membranes together with β2-AR, AC, Gαs, PKA, PP2A, and Cav-3. Caveolar membranes were fractionated from neonatal cardiomyocytes by homogenizing in sodium carbonate buffer and centrifugation to equilibrium in sucrose gradients. (A) One-milliliter fractions were collected from the top of the gradient and analyzed by SDS/PAGE and immunoblot analysis with antibodies to Cav1.2, KCNH2, AC, β1-AR, β2-AR, Gαs, Gαi, PKARII, PP2A, and Cav-3. (B) Protein recovery in each of the gradient fractions. Results are representative of data from six separate experiments.

Ravi C. Balijepalli, et al. Proc Natl Acad Sci U S A. 2006 May 9;103(19):7500-7505.
2.
Fig. 3.

Fig. 3. From: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ?2-adrenergic regulation.

Cav1.2 channels are associated with Cav-3 and components of β2-AR/AC/PKA signaling cascade in mouse hearts. Adult (A) and neonatal (N) mouse myocyte homogenates were subjected to immunoprecipitation with either anti-Cav1.2 or anti-Cav-3 antibodies, and the immunoprecipitates were analyzed by immunoblotting. Both Cav1.2 and Cav-3 are detected in the immunoprecipitates with either of the two antibodies, whereas control IgG does not immunoprecipitate the proteins (A), indicating an association between the two proteins. In B, immunoprecipitation of Cav1.2 or Cav-3 led to coprecipitation of AC, β2-AR, Gαs, PKARII, and PP2A but not β1-AR and KCNH2. A protein band for Gαi was detected only in the anti-Cav-3 immunoprecipitate. Results are representative of six different experiments.

Ravi C. Balijepalli, et al. Proc Natl Acad Sci U S A. 2006 May 9;103(19):7500-7505.
3.
Fig. 2.

Fig. 2. From: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ?2-adrenergic regulation.

Cav1.2 and Cav-3 are colocalized in adult canine and neonatal mouse ventricular myocytes. Isolated adult canine ventricular myocytes (A-C) and neonatal mouse myocytes (D-F) were immunolabeled with anti-Cav1.2 (A and D) and anti-Cav-3 (B and E) antibodies. Both proteins are detected on the surface membrane and in adult myocytes also in punctuate areas consistent with T-tubule localization. (C and F) Merged images with yellow regions indicating colocalization of Cav1.2 and Cav-3. (G and H) Immunogold colocalization of the Cav1.2 subunit of L-type Ca2+ channel (large particle, arrows) and Cav-3 (small particle, arrowheads) in the caveolae in isolated mouse cardiomyocytes. (Scale bars: 200 nm.).

Ravi C. Balijepalli, et al. Proc Natl Acad Sci U S A. 2006 May 9;103(19):7500-7505.
4.
Fig. 4.

Fig. 4. From: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ?2-adrenergic regulation.

Caveolar disruption with MβCD eliminated β2-AR but not β1-AR stimulation of ICa,L in neonatal mouse ventricular myocytes. Perforated patch whole-cell voltage clamp recordings of ICa,L were performed by using a holding potential of −40 mV with 50-ms test pulses to +20 mV every 15 s in myocytes pretreated with PTX. (A) Peak ICa,L was reversibly increased by β2-AR activation with 10 μM Salb plus 10 μM Aten and by β1-AR activation with 1 μM norepinephrine (NE) plus 1 μM prazosin (praz) in a representative cell (whole-cell capacitance = 13.3 pF). (B) Application of 10 mM MβCD eliminated the β2-AR but not β1-AR stimulation of ICa,L in a representative myocyte (whole-cell capacitance = 22.0 pF). (C) Average effect of β1-AR and β2-AR stimulation on ICa,L in cells with and without MβCD treatment. The number of cells tested is shown in parentheses. ∗, P < 0.005, MβCD-treated relative to control.

Ravi C. Balijepalli, et al. Proc Natl Acad Sci U S A. 2006 May 9;103(19):7500-7505.
5.
Fig. 5.

Fig. 5. From: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ?2-adrenergic regulation.

siRNA-mediated Cav-3 inhibition eliminated β2-AR stimulation of ICa,L in neonatal mouse ventricular myocytes. (A) Representative Western blot of Cav-3 and sarcomeric actin expression in myocytes with transfected with control and Cav-3 siRNA. (B) Densitometric analysis of Cav-3 expression normalized to sarcomeric actin (n = 5). Perforated patch whole-cell voltage clamp recordings of ICa,L were performed by using a holding potential of −40 mV with 50-ms test pulses to +20 mV every 15 s in myocytes treated with PTX. (C) Average current-voltage relationship of control siRNA (n = 6, ■) and Cav-3 siRNA (n = 6, ●). (D) Peak ICa,L is increased by β2-AR activation with 10 μM Salb plus 10 μM Aten in a representative control siRNA-treated myocyte (whole-cell capacitance = 7.7 pF). (E) siRNA-mediated Cav-3 inhibition eliminated β2-AR stimulation of ICa,L in a representative myocyte (whole-cell capacitance = 11.9 pF). (F) Average effect of β2-AR stimulation on ICa,L in myocytes with and without Cav-3 siRNA inhibition. ∗, P < 0.001 relative to control.

Ravi C. Balijepalli, et al. Proc Natl Acad Sci U S A. 2006 May 9;103(19):7500-7505.

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