Figure 7

siRNA-mediated knockdown of MMGL leads to reduced levels of cMyBPC phosphorylation under adrenergic stimulation. A. Graph demonstrating that real-time quantification of MMGL cDNA transcribed from RNA extracted from cells transfected with either a non-silencing control or a particular MMGL siRNA. Pde4dip Rn_RGD:708410_3_HP (MMGL 3) resulted in optimal knockdown of MMGL (80%). B. Western blots of 2-dimensional IEF gels showing the expression of the four phosphorylation isoforms of GFP-cMyBPC in H9C2 cells (i) under non-stimulated conditions; (ii) under adrenergic stimulation, (iii) under non-stimulated conditions in the absence of MMGL (i.e. with MMGL knock-down) and (iv) under adrenergic stimulation in the absence of MMGL. C. Quantification of cMyBPC isoforms in the autoradiographs of the 2-dimensional IEF gels shown in (B), graphing the levels of the four phosphorylation isoforms (0 = unphosphorylated; 1 = monophosphorylated, 2 = diphosphorylated, 3 = trisphosphorylated). B and C(i) show that under non-stimulated conditions, levels of the mono- and diphosphorylated forms are similar and increased compared to the trisphosphorylated form. B and C(ii) show that under adrenergic stimulation, there is a relative increase in the trisphosphorylated form of cMyBPC and reduction of the non-phosphorylated form. B and C (iii) show that under non-stimulated conditions, ratios of the four forms of cMyBPC are similar to that in wild-type cells. B and C(iv) show that, upon adrenergic stress, knock-down of MMGL leads to a reduction in the levels of all cMyBPC phosphorylation forms. Abbreviations: JL8 = antibody directed against GFP-MyBPC.