Fig. 1

Figure 1. CMG2 Interaction with the Actin Cytoskeleton (A) HeLa cells transiently transfected with cDNA expressing CMG2-V5 WT, D50A, or D50A with a deletion of the predicted CBD (D50A^ΔCBD) were lysed and CMG2 immunoprecipitated. IPs were then analyzed by SDS-PAGE and western blotting against actin and CMG2-V5. (B) HeLa cells were transiently transfected with cDNA expressing CMG2-V5 WT or D50A were FBS starved or not overnight, lysed and CMG2 immunoprecipitated. IPs were then analyzed by SDS-PAGE and western blotting against actin and CMG2-V5. (C) Control fibroblasts and fibroblasts from patient 1 carrying the D50N mutation. Cells were FBS starved or not overnight, lysed and CMG2 immunoprecipitated. IPs were then analyzed by SDS-PAGE and western blotting against actin and endogenous CMG2. (A–C) Actin was quantified by densitometry. Error bars represent standard error, n = 3; paired t test between D50A or D50N mutants and other constructs. n.s.: p > 0.05; ∗∗∗p < 0.001. (D) HeLa cells were transfected for 24 h with cDNA expressing CMG2 WT and plated on dishes coated with collagen I, collagen VI, or anthrax PA. After 1 h of adhesion, cells were lysed and CMG2 was immunoprecipitated. IPs were then analyzed by SDS-PAGE and western blotting against actin and CMG2-V5. (E) HeLa cells were transfected with siRNAs against talin (siRNA1 and 2), or vinculin for 72 h, and with cDNA expressing CMG2 WT for 24 h and finally serum starved overnight. Cells were then lysed, and CMG2 was immunoprecipitated. IPs were then analyzed by SDS-PAGE and western blotting against actin, vinculin, talin and CMG2-V5. (D and E) Representative experiment of n = 3. (F) Schematic representation of CMG2 interaction with the actin cytoskeleton. The cytoplasmic tail of CMG2 interacts with talin, which in turn interacts with vinculin, which is connected to actin.