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. Author manuscript; available in PMC: 2015 Mar 12.
Published in final edited form as: Nat Commun. 2014 Sep 12;5:4849. doi: 10.1038/ncomms5849

Figure 3. Shh and Smo increase Hhip protein turnover.

Figure 3

(a) HEK293T cells were transiently co-transfected with HA-Hhip (lanes 1–5), HA-HhipΔAR (lanes 6–10), and HA-HhipΔL2 (lanes 11–15) and the indicated Shh or Smo constructs. Western blot of whole cell lysates were immunoblotted with the indicated antibodies. (b) HEK293T cells were co-transfected with Hhip mutants and ShhC-HA, gD-SmoM2 or Shh. Western blot of whole cell lysates were immunoblotted with the indicated antibodies. Cotransfection of prk5-HA-Hhip and prk5-Shh (lanes 19–20) resulted in the same result as pcDNA3-HA-Hhip and prk5-Shh. Additionally, the Hhip AR region is involved in Hhip stability (lanes 10–12, 16–18). (c) HEK293T cells were transiently co-transfected with HA-Hhip and Ptch-HA or Shh. After 24 hours post transfection, cells were treated with water (control), 100 μM chloroquine, DMSO, 100 nM folimycin A, or 5μM MG132 for an additional 24 hours before lysis. For the Hhip HA immnunoblot, lanes 1–5 represent a shorter exposure than lane 6–10, the entire blot of both exposures is below. (d) As for panel c, cells were cotransfected with control vector or GLI3R. (e) Ptch−/− MEFs were cotransfected with HA-Hhip and indicated constructs, and 24 hours after transfection, cells were treated with 100 μM chloroquine for 4 hours. (f) HEK293T cells were transiently co-transfected with HA-Hhip (lanes 1–5), HA-HhipΔAR (lanes 6–10), or empty vector Control (lane 11–12) and either empty vector control, Smo-myc, or SmoΔCRD-myc and treated with 100 μM chloroquine. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody.