Fig. 2.

The association of HIF‐1α with NPM1 inside HeLa cells is regulated by ERK1/2. (A) Soluble proteins (INPUTS) or anti‐HIF‐1α IP of HeLa cells grown at 21% or 1% O₂ for 16 h, untreated (Ctr) or treated with 5 μm U0126 (+U0126) or deprived of serum (‐FBS) were analyzed by immunoblotting using antibodies against HIF‐1α, NPM1, phospho‐ERK1/2 and ERK1/2 as indicated. (B) Soluble extracts (INPUTS) or anti‐GFP IP of HeLa cells transiently expressing GFP or GFP‐tagged full‐length HIF‐1α WT, IA/SA, SE forms were analyzed at 20 h post‐transfection by immunoblotting using antibodies against HIF‐1α, GFP, ARNT, and NPM1 as indicated. (C) Immunofluorescence microscopy analysis of cells grown at 1% O₂ and treated as in (A) using antibodies against HIF‐1α (Green) or NPM1 (Red). Nuclei were stained with DAPI (Cyan; Scale bars: 10 μm). Middle panels are scatterplots of pixel intensities of HIF‐1α and NPM1 signals. Graph shows the Manders' overlap coefficient as measured in nuclei‐restricted fluorescence in 35 cells from two independent experiments in each condition ± SEM (***P < 0.001; Statistical variance between two groups of values was calculated by two‐tailed Student's t‐test). (D) Soluble proteins (INPUTS) or anti‐NPM1 IP of HeLa cells incubated at 1% O₂ for 16 h and treated (+U0126) or not (Ctr) with 5 μm U0126 were analyzed by SDS/PAGE and western blotting using antibodies against NPM1, HIF‐1α, and ARNT as indicated. (E) Soluble extracts (INPUTS) or anti‐GFP IP of HeLa cells transiently expressing GFP or GFP‐tagged full‐length HIF‐1α WT, S247A, S247D forms were analyzed at 20 h post‐transfection by immunoblotting using antibodies against HIF‐1α, GFP, ARNT, and NPM1 as indicated. Certain panels in A, B, D, E show single blot areas that correspond to the indicated molecular weight marker and were cut after blotting for analysis with different antibodies; images in A, B, D, E are representative of three (A) or two (B, D, E) independent experiments.