Figure 4

Rubicon-regulated \(\upbeta\)1 adrenergic receptor recycling under \(\upbeta\)1 adrenergic stimulation. (a) Western blot analysis of Rubicon in neonatal rat cardiomyocytes (NRCMs) infected with three different kinds of adenovirus expressing shRNA targeted to Rubicon mRNA (shRubicon). Right panel, quantitative analysis of Rubicon. Data were normalized to GAPDH and are expressed as fold increase over levels in NRCM infected with adenovirus expressing shRNA targeted to LacZ mRNA (shLacZ). N = 3. One-way ANOVA was used for analysis. *P < 0.0001 versus all the others. (b) Immunoblots for LC3. NRCMs infected with adenovirus expressing shLacZ or shRubicon were treated with or without 100 nM of Bafilomycin A1. Right panel, quantitative analysis of LC3-II. Data were normalized to GAPDH and the value for cells infected with shLacZ and treated with DMSO in each experiment was set equal to 1. N = 4. A one-way ANOVA was used for analysis. Open and closed bars indicate shLacZ and shRubicon, respectively. (c) Western blot analysis of \(\upbeta\)1 adrenergic receptor (AR) in NRCMs infected with shRubicon after isoproterenol treatment. Lower panel, quantitative analysis of \(\upbeta\)1AR. Data were normalized to GAPDH and are expressed as fold increase over levels in corresponding 0 h. N = 6. A non-repeated measure two-way ANOVA was used for analysis. Solid and dotted lines indicate significant differences between two different time points in shLacZ and shRubicon groups, respectively. Open and closed circles indicate, shLacZ and shRubicon #1, respectively. Cropped blots are displayed and full-length blots are presented in Supplementary Fig. S4. (d) Representative confocal images of recycling assay. Staining for \(\upbeta\)1AR is shown in green, and that for DAPI in blue. Arrows indicate NRCMs with weak green signal. Scale bar, 50 \(\upmu\)m. Right panel, quantitative analysis of \(\upbeta\)1AR-positive signal in cells. N = 4. Student’s t-test was used for analysis. Open and closed bars indicate, shLacZ and shRubicon, respectively. Data are expressed as the mean ± s.e.m. (e) Intracellular \(\upbeta\)1AR was analyzed by confocal microscope. Staining for \(\upbeta\)1AR is shown in green, and that for DAPI in blue. Scale bar, 50 \(\upmu\)m. Right panel, quantitative analysis of \(\upbeta\)1AR-positive signal in cells. N = 4. Student’s t-test was used for analysis. Open and closed bars indicate, shLacZ and shRubicon, respectively. Data are expressed as the mean ± s.e.m.