Clathrin localizes at centrosomes of interphase and metaphase cells. (A) HeLa cells were methanol fixed and processed for IF to detect CHC (X22 mAb) or CLC (rabbit polyclonal) and centrosomal markers PCNT2 (goat polyclonal) and γ-tubulin (mAb) at indicated cell cycle phases. Colocalization of green and red is shown as yellow. Bars, 10 µm. (B) HeLa-SNAP-uLCa clone 3.3 cells were methanol fixed and processed for IF to detect LCa with mAb X16 (CLC), PCM1 (rabbit polyclonal), and PCNT2 (goat polyclonal). Merged colors showing colocalization between labeled proteins are shown as yellow (red and green) and white (red, green, and yellow or cyan and red). Bars: (top) 2 µm; (bottom) 10 µm. (C) HeLa-SNAP-uLCa clone 3.3 cells were methanol fixed and processed for IF to detect TfR (mAb) or EEA1 (mAb), CLC with polyclonal rabbit antibody, and PCNT2 (goat polyclonal). Merged colors showing colocalization between CLC and PCNT2 are shown as pink. Bar, 10 µm. (A–C, insets) Boxes in each frame are magnified threefold. Bars, 2 µm. Images shown are 3D maximum projections.