Figure 4. Nestin prevents proteolytic processing of Gli3.
A, NIH3T3 cells were transfected with HA-tagged GFP, Gli3FL or Gli3R, and cell lysates were immunoprecipated with anti-Flag beads. Nestin, Actin and HA proteins in whole cell lysate, and precipitated Nestin were determined by western blotting. B, subcellular localization of exogenous Gli3FL or Gli3R in NIH3T3 cells transfected with HA-tagged Gli3FL or Gli3R, was examined by immunocytochemistry using anti-HA antibody. C, Cell lysates MB cells (isolated from 2 independent Math1-Cre/Ptch1C/C mice at 8 weeks of age) infected with a lentivirus carrying HA-tagged Gli3FL, were co-immunoprecipated with anti-HA beads. HA, Nestin and Actin proteins in the input and precipitated Nestin were examined by western blotting using designated antibodies. D, NIH3T3 cells transfected with HA-tagged Gli1, Gli2 or Gli3 in combination with Flag-tagged Nestin. Cell lysates were immunoprecipitated with anti-Flag beads. The presence of HA, Nestin and Actin proteins as well as precipitated Gli proteins were examined by western blotting using designated antibodies. E, Schematic representation of the GFP-tagged Nestin fragments. F, NIH3T3 cells were transfected with a HA-tagged Gli3 construct alone or together with GFP-tagged Nestin constructs. Nestin truncated proteins in the input and those precipitated with anti-HA beads were determined by western blotting using an anti-GFP antibody. G, NIH3T3 cells stably transfected with constructs carrying GFP (WT), Nestin (Nes O/E), or Nestin shRNA #4 (Nes null), were further transfected with HA-tagged Gli3FL. Whole cell lysates were immunoprecipitated with anti-HA beads, and phosphorylated Gli3 was examined by western blotting using an antibody recognizing phosphoserine.