Figure 6

In vivo co-immunoprecipitation of MMGL and its respective preys identified in the Y2H library screen. Western blots supporting the co-localization data in Figures 4 and 5. Antibodies used in immunoprecipitation (IP) and Western Blot (WB) are shown above each lane. Endogenous CARP (i), ENO1 (ii), ENO3 (iii) and cTNI (iv) immunoprecipitated exogenous dsRed-/YFP-MMGL in vivo in lysates of ds-Red-/YFP-MMGL transfected, differentiated H9C2 cardiomyocytes, while GFP-COMMD4 immunoprecipitated dsRed-MMGL in differentiated H9C2 cardiomyocytes transfected with both GFP-COMMD4 and dsRed-MMGL. Conversely, dsRed- or YFP-MMGL immunoprecipitated endogenous CARP (i), ENO1 (ii), ENO3 (iii) and cTNI (iv) in lysates of ds-Red- or YFP-MMGL transfected, differentiated H9C2 cardiomyocytes, while dsRed-MMGL also immunoprecipitated GFP-COMMD4. The dsRed antibody is directed against the dsRed-MMGL fusion protein, while the JL-8 antibody is directed against the YFP/GFP fusion proteins. The clear protein G control lanes show that these precipitations are not spurious, but are the result of physical association between the relevant proteins. Similar clear lanes were obtained when the HA antibody was used in negative control immunoprecipitation reactions (data not shown). Abbreviations: Prot G = protein G control; JL8 = antibody directed against YFP-tagged proteins, dsR = antibody directed against dsRed-tagged proteins, as described above.