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. Author manuscript; available in PMC: 2020 Sep 9.
Published in final edited form as: Dev Cell. 2019 Jul 3;50(5):610–626.e4. doi: 10.1016/j.devcel.2019.06.008

Figure 3. Hh induces Ci phosphorylation at C-I and C-II depending on Smo and Fu kinase activity.

Figure 3.

(A) The antigen sequences for generating the three phospho-specific antibodies.

(B-D) Western blot analysis of Myc-Ci−PKA and its variants in S2 cells co-expressed with FuEE and FuGV with the indicated phospho-specific antibodies.

(E) Western blot analysis of Flag-Ci−PKA phosphorylation in S2 cells with or without Sufu coexpression and treated with Hh-conditioned medium or control medium.

(F) Western blot analysis of Flag-Ci−PKA phosphorylation in S2 cells treated with the indicated dsRNA and Hh-conditioned medium and cotransfected without or with the indicated Smo or Fu construct.

(G) Western blot analysis of HA-Fu and HA-FuIns from S2 cells expressing the indicated Fu constructs and treated with GFP or Fu dsRNA.

(H-I) Western blot analysis of endogenous Ci phosphorylation from Cl8 cells treated with or without Hh-conditioned medium and the indicated dsRNA.

(J) Western blot analysis of endogenous Ci phosphorylation (top panels) or ptc-luc reporter assay (bottom panel) in Cl8 cells treated with increasing concentrations of Hh-conditioned medium.