Clathrin colocalizes with TACC3 and ch-TOG at interphase centrosomes, and ch-TOG levels are reduced by clathrin inactivation at S phase. (A) Asynchronous clone 3.3 cells were treated (2 h) with 5 µM BG-GLA-BG or DMSO (mock) and then methanol fixed and processed for IF to detect LCa (mAb X16), PCNT2, and ch-TOG. Three-color colocalization is white in the merged image, and red-blue colocalization is pink. Bar, 10 µm. (B) Asynchronous clone 3.3 cells were treated as in A, methanol fixed, and then processed for IF to detect LCa (rabbit polyclonal), PCNT2, and TACC3. Three-color colocalization is white in the merged image. Bar, 10 µm. (A and B, insets) Boxed regions are magnified threefold. Bars, 1 µm. Images are 3D maximum projections. (C) Clone 3.3 cells were enriched to S phase by T/T block and treated (2 h) at T0 with BG-GLA-BG or mock treated, as in A. Lysates prepared 4, 6, and 8 h after T/T were immunoblotted to detect ch-TOG, α-tubulin, and GAPDH, as indicated. Molecular mass marker position is shown on the right. (D) Clone 3.3 cells were enriched to S phase and treated with BG-GLA-BG or DMSO, as in C. Lysates prepared 2, 4, 6, 8, and 12 h after T/T were immunoblotted to detect TACC3 and GAPDH, as indicated. The immunoblot shown here comes from the same transfer membrane used to generate the immunoblot in Fig. S3 A. The transfer membrane was cut into horizontal strips of different molecular mass zones to detect all proteins shown here and in Fig. S3 A. Molecular mass marker position is shown on the right. (E) The densitometric quantification of immunoblots from multiple experiments performed as in C and D for clone 3.3 cells treated with BG-GLA-BG (shaded squares) or mock treated (open circles) relative to the loading control GAPDH. Data represent the mean ± SD of three to four separate experiments.
