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. 2009 May 8;284(19):12940–12948. doi: 10.1074/jbc.M900207200

FIGURE 5.

FIGURE 5.

Treatment of cells with the Hsp90 inhibitor 17-AGG and knockdown of Cdc37 reduce Ryk ICD levels in ESCs. A, Cdc37 binds to GFP-ICD, but not GFP-ICDΔ494–579 in ESCs. Prior to lysis, cells were treated with doxycycline (Dox) for 12 h on day 5 of culture growth. Mouse monoclonal anti-GFP immunoprecipitates (IP) were blotted with anti-Cdc37 antibodies or rabbit polyclonal anti-GFP antibodies. Un, undifferentiation conditions; Di, neural differentiation conditions. B, binding of Cdc37 to the Ryk ICD decreases during differentiation. Differentiating cells were cultured without doxycycline for various times (dashed line) and then treated with doxycycline for 12 h (red lines) to induce GFP-ICD expression. Each lysate was immunoprecipitated using anti-GFP antibodies followed by Western blotting with anti-Cdc37 and anti-GFP antibodies. C, the Hsp90 inhibitor 17-AGG affects GFP-ICD stability. After doxycycline treatment for the indicated times (red lines), Ryk-/- GFP-ICD ESCs were cultured with 17-AGG (1 μm) for 24 h (blue line). Western analysis with a GFP antibody shows a significant decrease in ICD levels at 36 and 72 h. Antibodies against Hsp90, Cdc37, and actin served as controls. D, a generation of ESCs inducibly expressing shRNA of Cdc37 is shown. A scheme of inducible expression of Cdc37 shRNA via tamoxifen (Tam)-induced recombination is shown in the top panel. Tamoxifen-induced recombination leads to reduction of mCherry signals (bottom left panel). Ryk+/+, Ryk-/- GFP, and Ryk-/- GFP-ICD ESCs infected with constructs expressing Cdc37 shRNA and Cre-ERT were treated with vehicle or tamoxifen and lysed after three passages. Western blot analysis confirms tamoxifen-regulated Cdc37 knockdown (bottom right panel). Ub, ubiquitin. E, GFP-ICD stability is reduced by Cdc37 knockdown. Ryk-/- GFP and Ryk-/- GFP-ICD ESCs expressing the empty construct or encoding Cdc37 shRNA were cultured under undifferentiation or neural differentiation conditions, incubated with doxycycline for the indicated time (red line) to induce GFP or GFP-ICD, and analyzed by Western blotting using antibodies against the indicated proteins.