Prohibitin2, a novel mDia1-interacting protein, associates with mDia1 in myotubes. (a) Domain structure of full-length (FL) mDia1 and constitutively active mDia1 mutant, mDia1ΔN3. 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. Start 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 mDia1ΔN3-BD (positive GAL4 reconstitution) or empty-BD (negative 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. Positive control “P”- Drosophila Batman-AD and GAGA factor-BD, negative control “N”- empty-AD and empty-BD. Trp-Tryptophan, Leu-Leucine, Ade-Adenine. AD-Activation domain, BD-binding domain. (c) Domain structure of Phb2 FL and Phb2-Y2H. HYD-Hydrophobic region, PHB-Prohibitin domain, CC-Coiled coil domain. (d) Co-IP of flag-tagged Phb2-Y2H and GFP-tagged mDia1ΔN3 to confirm the interaction. HEK293T, co-transfected with mDia1ΔN3 and Phb2-Y2H, and pulled down with anti-Flag antibody. IP product was run on two different gels 8% and 12% for detecting with anti-GFP and anti-Flag antibodies respectively and these blots were processed in parallel. The blot probed with anti-Flag antibody represented here was cut prior to processing for western blotting. Cropped blot for GFP has been shown here whereas full-length GFP blot is presented in Supplementary Fig. S6. (e) LC-MS/MS analysis of mDia1-interacting proteins in myoblasts (MB) and myotubes (MT) in differentiation medium (DM) for 72 hours. Venn diagram represents the number of proteins that bind mDia1 in MB or MT or both MB and MT. (f) Phb2 peptides identified in MT lysates by LC-MS/MS analysis of mDia1 IP proteins. Phb2 aa sequence (NCBI Reference Sequence # NP_031557.2) showing peptides identified in first (red), second and third (blue) and all three (underlined) biological replicates. (g,h) Reciprocal IP of endogenous mDia1 and Phb2 to identify stage-specific interaction. Lysates from proliferating MB (GM), MT in DM for 24 (D24) and 72 (D72) hours were harvested and subjected to IP with anti-mDia1 (g) or anti-Phb2 (h) antibodies. IP samples were loaded on different gels, blots were cut and processed in parallel, using same conditions of antibody incubation and exposure time during developing. (i) Western blot showing the expression profile of mDia1 and Phb2 in GM, D24, and D72 lysates. All the lysates were run on a single gel, blots were cut and probed for mDia1, Phb2, Akt2, MyoD, β-actin and GAPDH. The same lysates were run on a different gel, blots were cut and probed for MyoG, Akt1 and GAPDH. (j,k) Bar diagram represents the densitometric quantification of western blots shown in (i) *p < 0.05, **p < 0.01, ***p < 0.001 when compared to GM, n = 3. a.u. -arbitrary units. Numbers represent aa position (a,c). Corresponding sizes are indicated in kDa. ns-not significant.