Figure 4.

FTS, Hook proteins, and p107^FHIP form a single major complex as assessed by gel filtration. (A) Effect of Hook1 depletion on the abundance of Hook2 and Hook3 proteins in complexes with FTS. Extracts were made from HEK293T/FLAG-HA-FTS cells transfected with the indicated siRNAs and subjected to immunoprecipitation with anti-HA antibodies. Blots were probed with the indicated antibodies. (B) Gel filtration analysis of FTS/Hook complexes in wild-type cells and in cells depleted of either FTS or Hook proteins. HeLa cells were transfected with the indicated siRNAs for 48 h before lysis (see Supplemental Figure S2 for depletion). Extracts were subjected to gel filtration by using a Superdex 200 column as described in Materials and Methods (B). Aliquots of fractions were analyzed by immunoblotting using the indicated antibodies. Anti-FTS antibodies were affinity purified. (C) Gel filtration analysis of FLAG–HA–FTS complexes purified from HEK293T cells. Complexes purified as described in Figure 1 were subjected to gel filtration using a Superdex 200 column, and fractions were analyzed by immunoblotting. (D) LC/MS/MS analysis of purified FLAG–HA–FTS complexes after gel filtration. FLAG–HA–FTS complexes purified as described in Figure 1 were subjected to gel filtration as described in B. Fractions corresponding to the peak of Hook/FTS proteins by immunoblotting (C) were precipitated with trichloroacetic acid, trypsinized, and subjected to LC/MS/MS analysis to identify p107^FHIP. The number of unique peptides and total scans for the relevant proteins are indicated.