Fig. 5: Characterization of LDB3 interactions with filamin C and HSPA8.
a Representative immunoblots showing protein levels of filamin C, BAG3, and HSPA8 relative to β-actin in the vastus muscle of 8-month-old Ldb3Ala165Val/+ mice and Ldb3+/+ littermates. Data represent n = 5 mice per group and triplicate assays. b Schematics of LDB3 interaction with the mechanosensing domains Ig17–21 of filamin C and its chaperone HSPA8 as identified by yeast two-hybrid (Y2H) screen of a human skeletal muscle cDNA library. See Supplementary Table 1. Locations of the LDB3 bait encoded by exons 8-11Δ10 (yellow) and the p.Ala165Val mutation (white; asterisk) within actin-binding domain (ABD; blue) are shown in LDB3-LΔex10 isoform7,17. Domain composition of the prey clones are shown. c Pairwise Y2H assays demonstrating interaction between LDB3 peptides and HSPA8. Positive interactions show yeast growth on the media deficient in HIS3 and ADE2. Yeast cells co-transformed with empty bait vector and the HSPA8 prey show no growth (labeled –). Transformation efficiency was uniform for all constructs. Sequential tenfold yeast dilutions are shown. d GST pulldown assay shows that GST-tagged wildtype (WT) and mutant LDB3-LΔEX10 (Ala165Val) but not GST alone pulled down filamin C and its interactor the CASA cochaperone BAG3 from the vastus muscle lysates of wildtype mice. Data represent n = 3 mice and triplicate assays. e Co-immunoprecipitation (co-IP) assays show that a FLAG antibody pulled down the FLAG-tagged WT and mutant (Ala165Val) LDB3-LΔex10 together with HA-tagged filamin C rod domain Ig17–21 and HSPA8 in Cos7 cells. Data represent triplicate assays. The proteins were detected with anti-FLAG and anti-HA antibodies. f Co-IP assays show that an LDB3 antibody pulled down LDB3 isoforms together with filamin C and HSPA8 from the tibialis anterior muscle lysates of Ldb3Ala165Val/+ and Ldb3+/+ mice. Data represent n = 3 mice per group and triplicate assays.
