Figure 2.

Overall protein O-GlcNAc levels during various cell cycle phases. (A) HeLa cells were synchronized and allowed to recover from the thymidine blockage. The number of mitotic cells at the indicated times was assessed as the number of round shaped cells per field of view using bright-field microscopy. Data are shown as means ± SD from at least 3 independent experiments; (B) representative Western blot analysis using RL2 or CTD110.6 anti-O-GlcNAc antibody and anti-actin antibody on total protein extracts isolated from synchronized cells. Cells were collected at specific cell cycle phases as described in the Materials and Methods. For the mitotic cells, we pooled the first six fractions (M1) and the last three fractions (M2) prior to lysis; (C) densitometric analysis of CTD110.6 staining of whole lanes in the four main cell cycle phases (top panel) and densitometric analysis of RL2 staining of whole lanes and three individual bands (at ~150, 100 and 60 kD, as indicated by arrows in (B) in the four main cell cycle phases. Data from M1 and M2 fractions were combined for statistical analysis. Data are shown as means ± SD, * p < 0.05 vs. G₁.