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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Mol Cell Neurosci. 2012 Apr 2;50(1):82–92. doi: 10.1016/j.mcn.2012.03.008

Figure 2.

Figure 2

Shh preferentially activates the PKCα isoform. A, Chick retinal explants were treated with 3.0 μg/ml Shh for 5 and 15 min. Cell lysates were processed to result in cytosolic, Triton X-100 soluble and insoluble fractions. Western blots were carried out by using antibodies specific for various PKC isoforms and loading controls were shown blotted with GAPDH, Na+/K+-ATPase and α-tubulin antibodies. Representative gel image of three independent experiments is shown. B, Quantification of translocation of PKC isoforms to TritonX-100 soluble and TritonX-100 insoluble fractions. C, Chick retinal explants were treated with vehicle control, Shh alone or Shh in the presence of Gö6976 or PKCβ inhibitor for 5 mins. Western blots were carried out by using antibodies specific for phospho-PKCα (S657) or phospho-PKCβI (T642). Representative gel image of three independent experiments is shown. C′, Quantification of phospho-PKCα (S657) elevation in chick retinal explants treated with vehicle control, Shh alone or Shh in the presence of Gö6976 or PKCβ inhibitor for 5 mins. Data are represented as mean±SEM. *p<0.05, Student’s t test. D, E, Chick RGC axon cultures were treated with vehicle control, Shh alone or Shh in the presence of Gö6976 or PKCβ inhibitor for 2 mins. Representative immunofluorescent images of antibody staining specific for phospho-PKCα (S657) are shown. The amount of fluorescence in each distal axon was quantified by ImageJ (see Methods) and the mean values were calculated. Numbers in parentheses indicate the total number of axons measured from three independent experiments. F, Fluorescent images of Fluo-3 AM-loaded growth cone at 0″, 33″ and 104″ after the onset of Shh application from a micropipette positioned at ~150 μm from the growth cone. G, Average Fluo-3 fluorescence changes in the whole growth cones after application of vehicle (n=7), Shh (n=7) or Shh with cyclopamine (n=6) were depicted as percent changes normalized to the average fluorescent intensity before the onset of vehicle or Shh application (time 0) (ΔF/F). Data are represented as mean±SEM. *p<0.01, Student’s t test.