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. 2009 Oct 15;4(10):e7471. doi: 10.1371/journal.pone.0007471

Figure 7. Phosphorylation of GLI3.

Figure 7

(a) Left: GLI3-Flag was expressed in HeLa cells and isolated from nuclear or cytosolic fractions by immunoprecipitation. Immunoprecipitates were analyzed by Western blot. Phosphorylation of GLI3-Flag was visualized by staining with a pool of phospho-serine and phospho-threonine antibodies (upper panel). Afterwards the same membranes were stripped and incubated with Flag-antibodies to verify that the phospho-specific bands co-stain with GLI3-flag (lower panel). Right: To control the efficiency of the fractionation procedure, aliquots of the nuclear and cytosolic fractions were analyzed on Western blots with anti-LaminA/C- and anti-Tubulin-antibodies. (b) Nuclear and cytosolic fractions of GFP-GLI3-AA568-1549 over-expressing HeLa cells were separated on 2D-Gels and blotted with an anti-GFP-antibody. (c) Different concentrations of the acrylamide-pendant Phos-tag ligand (0, 5, 10, 20, 40 µM) have been added to 6% SDS-Gels that were used to analyse GFP-GLI3, wild-type (wt) and mutant (A934P, I808M, P707S). An anti-GFP antibody has been used for detection.