Fig. 2. GBP and Frat associate with kinesin light chain (KLC) in vivo. (A) KLC1 and Frat1 co-immunoprecipitate in COS-7 cells. Cells were transfected with full-length FLAG-tagged mouse Frat1 (mFrat1) and a fragment of mouse KLC1 obtained in the two-hybrid screen that consists of the N-terminal 198 residues of KLC1 fused to a Glu-Glu epitope tag (mKLC1). Cell extracts were immunoprecipitated with anti-GG or anti-FLAG antibody as indicated (IP), and the lysates were analyzed by Western blot as indicated (Blot). A portion of the cell lysate was reserved before immunoprecipitation and analyzed by Western blot to confirm expression of the transfected constructs (TL). Lanes 3 and 7 show the level of background binding of mFrat1-FLAG and mKLC1-GG, respectively, to the protein A/G beads used for immunoprecipitation. (B) XKLC4 co-immunoprecipitates GBP in Xenopus embryos. Embryos were injected with XKLC4-HA and GBP-myc RNAs at the 2-4-cell stage, lysed after 4 hours and immunoprecipitated (IP) with anti-HA antibody or no antibody (no Ab) as a negative control (left panel). A portion of each sample was taken prior to immunoprecipitation to show expression of injected RNAs (total lysates, right panel). Samples were analyzed by Western blot with anti-myc and anti-HA antibodies (Blot).