Vered within the Y2H screen (Fig. 1c), was applied for co-immunoprecipitation (IP) to validate the mDia1-Phb2 interaction in mammalian cells. IP with anti-flag in HEK293T cells co-expressing GFP-tagged mDia1N3 and Phb2-Y2H, resulted in co-IP of mDia1N3 (Fig. 1d), confirming that ectopically expressed mDia1 and Phb2 can interact in mammalian cells.Identification of novel interacting partners of mDia1 reveals Phb2, a multi-functional transcriptional regulator. Previously we showed that mDia1 regulates the expression of MyoD in proliferating MB, byLC-MSMS evaluation of mDia1-interacting Sunset Yellow FCF custom synthesis proteins in MB and MT. To assess the range of mDia1-interacting proteins in muscle cells, we performed LC-MSMS analysis of mDia1-co-IPs from two stages: proliferating MB and MT in differentiation medium (DM) for 72 hours. mDia1 was identified in both MB and MT, confirming thriving immunoprecipitation from both states (Supplementary Table S2). Notably, Phb2 was identified as an mDia1-interacting protein specifically in MT in all three replicates (Supplementary Table S4), additional validating the mDia1-Phb2 interaction noted in the Y2H analysis. Phb2 peptides identified by mass spectrometry are shown in Fig. 1f. 13 proteins had been typically associated with mDia1 in each MB and MT. 11 more mDia1-interacting proteins have been exclusively detected in MB and 104 were identified only in MT (Fig. 1e). These proteins had been reproducibly detected in 3 independent biological replicates of endogenous mDia1 IP-LC-MSMS analysis. mDia1-interacting proteins prevalent to MB and MT and certain to MB or MT are listed in Supplementary Tables S2 four respectively. We made use of REVIGO45 for gene ontology (GO) evaluation of mDia1-interacting proteins (Supplementary Fig. S2). The altered interactome inside the two stages suggests that mDia1 function might differ through myogenesis, with an expanded function in MT. STRING analysis of mDia1-interacting proteins identified clusters of interacting proteins in both states (Supplementary Fig. S3). Networks of mDia1-interacting proteins popular to MB and MT or particular to either MB or MT are shown, highlighting, MT-specific networks of proteasomal proteins (red), metabolic enzymes (blue) and mitochondrial proteins (black). The altered mDia1 interactome suggests stage-specific changes in effector function through myogenesis. Since Phb2 has been previously implicated in myogenic differentiation42,43, we delineated the consequences of its interaction with mDia1 in detail.Scientific RepoRts | (2019) 9:8302 | 41598-019-44749-www.nature.comscientificreportswww.nature.comscientificreportsFigure 1. Prohibitin2, a novel mDia1-interacting protein, associates with mDia1 in myotubes. (a) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1N3. Grey lines indicate RhoA and DAD binding regions. G-GTPase binding domain, DID-Diaphanous Inhibitory Domain, Dimerisation Domain (DD), Coiled Coil (CC), FH1, FH2, FH3-Formin Homology domains, DAD-Diaphanous Auto-inhibitory Domain. Begin positions of domains are depicted. (b) Phb2 identified as mDia1-interacting protein in a yeast two-hybrid screen. PJ69-4A was co-transformed with Phb2-AD and mDia1N3-BD (constructive GAL4 reconstitution) or empty-BD (damaging GAL4 reconstitution) and four colonies per reconstitution were screened for ADE2 and LacZ reporters on -Trp-Leu-Ade and -Trp-Leu + X-Gal plates respectively. Growth indicates ADE2 induction and blue pigmentation indicates LacZ induction. Optimistic handle “P”Droso.