Figure 4.

PKM2 interacts with HuR in a pY-dependent manner to control p27 levels and cell cycle progression. (a)Western blot analysis of levels of FLAG and HuR in FLAG immunoprecipitates from control or PKM2 shRNA-expressing U87 and LN319 cells expressing FLAG-tagged mouse WT (mM2) or R399E, K367M or K433E forms of PKM2. Western blot analysis of total PKM2 (bottom of panel) was used to verify equal input. (b) Western blot analysis of levels of p27, HuR, PKM2 and bactin in total cell lysates from control or PKM2 shRNA 1-expressing U87 (left) and LN319 (right) cells expressing WT (mM2) or R399E, K367M, or K433E forms of PKM2, or PKM1. See also Supporting Information Figures S4a–S4e. (c) Western blot analysis of pY and HuR levels in FLAG immunoprecipitates from U87 cells expressing WT Y200F FLAG-tagged HuR and a scramble or PKM2-targeted shRNA. (d)Western blot analysis of HuR in the FLAG immunoprecipitates from the nuclear (N) and cytoplasmic (C) fractions of control or PKM2 shRNA-containing U87 and LN319 cells expressing FLAG-tagged WT (left) or Y200F (right) HuR. DAPI (blue)/FLAG (green) co-immunofluorescence analysis was used to verify the Western blot assignment of sub-cellular fractionation. PKM2 staining (red) delineates the cytoplasm in groups with minimal cytoplasmic HuR. (e) Western blot analysis of FLAG, PKM2 and HuR in the FLAG or IgG immunoprecipitates from U87 and LN319 cells expressing FLAG-tagged WT or Y200F HuR. (f,g)Western blot analysis of p27 and b-actin (f) and cell cycle analysis (g) in control and PKM2 shRNA-expressing U87 and LN319 cells also expressing WT or HuR Y200F-encoding, FLAG-tagged constructs. *,p<.05,n = 3