Fig. 3

Fig. 3 Figure 3. CMG2 Interaction with the Talin F3 Domain (A) MD simulation of the complexes modeled for CMG2 CBD bound to the talin F3 domain (PDB 3G9W). The set of interactions between residue W396 of CMG2 and the talin F3 domain was held constant during the simulation. Residues mutated in HFS patients are highlighted in red. The talin interaction motif consisting of K399, G400 and S401is highlighted in blue. (B) Based on the sum of the interactions found in four independent restrained simulations, an average number of interactions that each amino acid of CMG2 CBD makes with any amino acid of the talin F3 domain was determined. The values were normalized as a percent of the number of interactions observed for W396. The color code is similar as in (A). (C) HeLa cells were transfected for 24 h with cDNA expressing CMG2-V5 WT, D50A, Y380A and Y381A and the cells were grown with or without FBS overnight . CMG2 was then immunoprecipitated, and IPs were analyzed by SDS-PAGE and western blotting against talin, vinculin, actin and V5. (D) HeLa cells were transfected for 24 h with cDNA expressing CMG2-V5 WT, G385R and G385A and the cells were grown without FBS overnight and treated with 500 ng/ml of PA at 4°C for 1h and switched to 37°C for 10 minutes before lysis. CMG2 was then immunoprecipitated, and IPs were analyzed by SDS-PAGE and western blotting against talin, vinculin, actin, PA, and V5. (E) HeLa cells were transfected for 24 h with cDNA expressing CMG2-V5 WT, V386G and V386A and the cells were grown without FBS overnight and treated with 500 ng/ml of PA at 4°C for 1h and switched to 37°C for 10 minutes before lysis. CMG2 was then immunoprecipitated, and IPs were analyzed by SDS-PAGE and western blotting against talin, vinculin, actin, PA, and V5. (F) HeLa cells were transfected for 24 h with cDNA expressing CMG2-V5 WT, D50A, K399A, G400A and S401A and the cells were grown with or without FBS overnight . CMG2 was then immunoprecipitated, and IPs were analyzed by SDS-PAGE and western blotting against talin, vinculin, actin and V5.