Figure 1. Cell cycle-dependent localization of UNC119a. Asynchronously growing HeLa cells were fixed in cold methanol (A and B) or paraformaldehyde (D). Fixed cells were double immunostained with UNC-Ab and monoclonal α-tubulin-Ab (or monoclonal γ-tubulin-Ab). The binding of the antibodies was visualized using Alexa 546-conjugated goat anti-rabbit IgG (red) and Alexa 488-conjugated goat anti-mouse IgG (green), respectively. The nuclei and chromosomes were stained with DAPI. (A) Cell cycle-dependent localization of UNC119a. White arrows show regions of highly concentrated UNC119a. Bars: 5 μm, in interphase; 10 μm, in other images. (B) UNC-Ab specifically recognizes endogenous UNC119a. Fixed HeLa cells were double immunostained with monoclonal α-tubulin-Ab and UNC-Ab pre-incubated with GST-UNC119a (UNC + UNC Ag). Pre-incubation of UNC-Ab with the antigen completely inhibited the binding of UNC-Ab to the endogenous protein. Bars, 20 μm. (C) The cellular content of UNC119 does not change during the cell cycle. HeLa cells were synchronized at G₁/S using a double thymidine block. Synchronized cells were released into fresh medium, collected at the indicated times and lysed with RIPA buffer. Western blotting was performed with three antibodies: UNC-Ab for UNC119a; anti-phosphor-histone H3 (Ser 10)-Ab, pHH3 (S10), as a mitosis marker; and monoclonal α-tubulin-Ab as a loading control. A.S., asynchronous cell extracts. (D) Cell cycle-dependent localization of UNC119a in paraformaldehyde-fixed cells. Asynchronously growing HeLa cells were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS. Double immunostaining was performed as described above. White arrows indicate the centrosomes. Bar, 20 μm.