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. 2007 Sep 24;27(23):8178–8189. doi: 10.1128/MCB.00656-06

FIG. 5.

FIG. 5.

Mad3 is required for Shh-mediated GNP proliferation. (A) Anti-HA (top) or anti-Flag (bottom) immunoblot of protein extracts from HEK293 cells expressing HA-Mad3 or Flag-Nmyc with pEGFP-Con-shRNA or pEGFP-Mad3-shRNA or with Nmyc-siRNA or control-siRNA, respectively. (B) Granule cells were transfected with pEGFP-Con-shRNA or pEGFP-Mad3-shRNA and cultured for 2 days in the presence or absence of Shh. During the final 12 h of culture, cells were pulsed with BrdU. Cells were fixed and stained with anti-EGFP and anti-BrdU antibodies. (C to E) Graphs depict the percentage of transfected cells (EGFP+) that expressed Mad3 (C), incorporated BrdU (D), or expressed Nmyc (E) in PBS (black)- or Shh (gray)-treated cells upon Mad3 knockdown compared with control shRNA. (F) The graph depicts the percentage of transfected cells (EGFP+) that expressed Mad3 in PBS (black)- or Shh (gray)-treated cells upon Nmyc knockdown compared with a control siRNA. Error bars represent standard errors of the means for three or four wells; each well contained 25 to 50 transfected cells. Similar results were obtained in three independent experiments. Asterisks indicate P values of <0.05 for comparisons indicated with black lines. Bar, 20 μm.