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. 2019 Nov 15;12(11):dmm041103. doi: 10.1242/dmm.041103

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© 2019. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 6. — Altered binding of titin and MURF1 caused by the TTN mutations. (A) GFP-tagged TTN-A168-170 domain co-immunoprecipitated with myc-tagged MURF1 is shown (top panel). Expression levels of GFP-tagged TTN-A168-170 (middle panel) and myc-tagged MURF1 (lower panel) were evaluated by WB of whole-cell lysates. Binding pairs were WT or D30994N mutant TTN-A168-170 in combination with MURF1. Dashes indicate no GFP-tagged or myc-tagged proteins (transfected only with pEGFP-C1 or pCMV-Tag3, respectively). (B) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of precipitate normalized to the input amount of GFP-TTN. Value for WT TTN-A168-170 with MURF1 was arbitrarily defined as 1.00 AU. *P<0.01 versus WT. (C) GFP-tagged TTN-A168 or TTN-A169 domains co-immunoprecipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP-tagged TTN-A168 or TTN-A169 (middle panel) and myc-tagged MURF1 (lower panel) were evaluated by WB of whole-cell lysates. Binding pairs were WT or D30994N mutant TTN-A168 in combination with MURF1, and WT TTN-A169 in combination with MURF1. (D) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of precipitate normalized to the input amount of GFP-TTN. Value for WT TTN-A168 with MURF1 was arbitrarily defined as 1.00 AU. Data are represented as means±s.e.m. (n=10). *P<0.01 versus WT. (E) GFP-tagged TTN-A160-161 domains co-precipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP-tagged TTN-A160-161 (middle panel) and myc-tagged MURF1 (lower panel) was confirmed by WB of whole-cell lysates. Binding pairs were WT or S30186A mutant TTN-A160-161 in combination with MURF1. Dashes indicate no myc-tagged proteins (transfected only with pCMV-Tag3). (F) Densitometric data obtained from the co-IP assays. Bar graph indicates the amount of IPs normalized to the input amount of GFP-TTN. Value for WT TTN-A160-161 with MURF1 was arbitrarily defined as 1.00 AU. Data are represented as means±s.e.m. (n=10). **P<0.001 versus WT. (G) Titin A168-170 was ubiquitinated by MURF1 and MURF2. The titin fragment A168-170 containing the MURF1-binding site was used as a substrate for in vitro ubiquitination assays. Addition of recombinant MURF1 or MURF2 catalyzes multi-ubiquitination of titin fragments. Lanes: Co, control (no E3 ligase added); M1, inclusion of MURF1; M2, inclusion of MURF2. After incubation, A168-170 fragments were detected by WB, using specific antibodies to titin A169 (left), and A168-170 (right).