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

Fig. 3.

Fig. 3.

The mutant TTN Ig domain binds more strongly to MURF1 and is degraded faster than wild type, both in vitro and in vivo. (A) RT-PCR analysis of TTN for the N-terminal portion, A-I junction region, exon 204 and titin kinase domain in the WT and nsh mutant embryos at 3 dpf. Despite the presence of a missense mutation, the level of TTN mRNAs for these portions in the nsh mutant was similar to that in the WT. The ef1α transcript was used as a control. (B) RT-PCR analysis of titin N2B isoforms in the WT and nsh mutants. N2B isoforms were increased. (C-F) Embryonic skeletal muscle of the WT and nsh mutant at 3 dpf were stained with antibodies to A-I junction titin (C,D) and C-terminal titin (E,F). Despite the similar expression level of TTN mRNA, expression of titin containing the A-I junction isoform was reduced (D). Sarcomeric structures were disrupted in the nsh mutant (F). (G-J) WT and mutant (E29783V which was the equivalent of D23186V mutation in medaka) Ig fragments mRNA injection into medaka embryos. Despite the same amount of mRNA injection, mutant (E29783V) Ig fragments degraded faster than WT TTN Ig fragments. (K) Western blots (WBs) of HEK293FT cells transfected with HA-tagged WT and mutant (E29783V) Ig fragments with or without a proteosome inhibitor, epoxomicin. Myc-tagged ubiquitin was co-transfected to visualize ubiquitination. WBs of anti-HA IPs and control normal IgG IPs are shown. Note that the myc-labeled large molecules in mutant TTN transfectants were present only in the presence of epoxomicin. (L) GFP-tagged TTN-A156 domain co-precipitated with myc-tagged MURF1 are shown (top panel). Expression levels of GFP-tagged TTN-A156 (middle panel) and myc-tagged MURF1 (lower panel) was confirmed by WB of whole-cell lysates. Binding pairs were WT or E29783V mutant TTN-A156 in combination with MURF1. Dashes indicate no myc-tagged proteins (transfected only with pCMV-Tag3). (M) Densitometric data obtained from the co-IP assays. Bar graphs indicate the amount of IPs normalized to the input amount of GFP-TTN. Value for WT TTN-A156 with MURF1 was arbitrarily defined as 1.00 arbitrary unit (AU). Data are represented as means±s.e.m. (n=10). **P<0.001 versus WT. Scale bars: 50 µm.