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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Circ Heart Fail. 2014 Feb 13;7(3):479–490. doi: 10.1161/CIRCHEARTFAILURE.113.000713

Figure 4. MuRF1 plays a critical role in mediating ubiquitination of CnA.

Figure 4

A, Left: Heart homogenates were incubated with agarose-conjugated GST-TUBE2 to obtain ubiquitinated proteins. The samples were then subjected to immunoblot analyses with anti-CnA antibody. Right: An immunoblot of the input with anti-ubiquitin antibody. The results are representative of three experiments. B, Heart homogenates were subjected to immunoprecipitation with anti-CnA antibody or control IgG. The immunoprecipitates were immunoblotted with anti-MuRF1 antibody. C, Heart homogenates were subjected to immunoprecipitation with anti-MuRF1 antibody or control IgG. The immunoprecipitates were immunoblotted with anti-CnA antibody. D, Cultured cardiomyocytes were transduced with either Ad-sh-MuRF1 or Ad-sh-scramble. Ninety-six hours after transduction, cells were treated with cycloheximide (CHX) and then harvested after the indicated times. Immunoblot analysis was performed using anti-CnA antibody. The amount of CnA was normalized with that of α-tubulin at each time point and expressed as a percentage of the value at time 0 h. * P<0.05 vs. Ad-sh-scramble at that time point (N=3). E, In vitro ubiquitination reactions were performed to test the ubiquitin ligase activity of MuRF1. Ubiquitin conjugates of CnA were detected with anti-CnA (left panel) and anti-ubiquitin antibodies (right panel). The ubiquitination of CnA is represented by the accumulation of slower-migrating species of CnA with concomitant loss of the nonubiquitinated protein. The results are representative of three experiments. F, ZAKI-4β abrogates the enhancement of TAC-induced increases in the CnA activity in Murf1−/− mice. Tg-NFAT-Luc mice were crossed with Tg-ZAKI, Murf1−/−, Murf1−/−-Tg-ZAKI cross or control mice, and each mouse group was subjected to either sham or TAC operation. After one week, NFAT-Luc activity in the heart homogenates was evaluated. * P<0.05 (N=4–7). G, Calcineurin phosphatase activity in the hearts of Tg-ZAKI, Murf1−/−, Murf1−/−-Tg-ZAKI or NTg mice subjected to either sham or TAC operation. * P<0.05 (N=6).