Mapping interaction domains on mDia1 and Phb2. (a) Schematic for mouse mDia1 truncation mutants. (b) Western blot to detect expression level of mDia1 mutants. Lysates from HEK293T transfected with mDia1 mutants were probed using anti-GFP antibody. GAPDH was used as a loading control. All the lysates were run on a single gel, the blot was cut and processed in parallel for detection of GFP-tagged mDia1 mutants and GAPDH. (c,c’) Co-IP of mDia1 mutants and Phb2 to map interaction domains. HEK293T cells were transfected with Phb2-Y2H and various mDia1 mutants, followed by IP with anti flag antibody. IP products were run on different gels, the blots were cut and processed in parallel for detection of interacting mutants. (d) Schematic for mouse Phb2 truncation mutants. (e) Western blot to detect expression of Phb2 mutants. Lysates of HEK293T transfected with Phb2 mutants were loaded on a single gel, the blot was cut and analysed in parallel using anti-flag and anti-GAPDH antibodies. GAPDH was used as a loading control. (f) Co-IP of Phb2 mutants and mDia1 to map interaction domains. Lysates from HEK293T cells co-transfected with various Phb2 mutants and mDia1ΔN3 were subjected to IP using anti-flag antibody. IP samples were run on different gels, the blots were cut and processed in parallel for detection with anti-GFP and anti-Flag antibodies under same conditions of detection. Different gels with 12% and 8% were used for the flag and GFP blots respectively. The represented cropped input lanes of Phb2-Amino, Central, Carboxy and 120–232 in the GFP blot show lower exposure of the inputs run along with the corresponding IP samples in the same gel whereas the cropped input lanes from Central, Carboxy and 120–232 in flag blot represent higher exposure of the inputs run along with the corresponding IP samples in the same gel. The numbers represent aa positions (a,d).