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Fig. 6 Hhip and Smoothened colocalize at the cell surface and internalize together with Clathrin in response to Hh. (A–F) Hhip and Smo, but not Ptc, colocalize at the cell surface. COS7 cells transfected with myc-Hhip, flag-Smo or Ptc-HA. (A–C) Hhip is located on the cell surface (A, arrow) and intracellularly. Intracellular Hhip localizes with Calreticulin and GM130, markers of endoplasmic reticulum and Golgi, respectively (Supplementary Fig. 4). Myc-tagged Hhip (A), Flag-tagged Smo (B) and merged image (C). (E) Ptc is located predominantly intracellularly. Myc-tagged Hhip (D), HA-tagged Ptc (E) and merged image (F). (G–X) Hhip and Smo internalize together with Clathrin in response to Hh. COS7 cells transfected with myc-Hhip, flag-Smo or Ptc-HA and then incubated for 2 h with control medium (G–I, M–O, S–U) or ShhN conditioned medium (J–L, P–R, V–X). (G–R) Hhip and Smo distribute to juxtanuclear and peripheral vesicular structures. (M–R) Ptc does not colocalize with Hhip (R, arrow). (S–X) Hhip localizes with endogenous Clathrin in ShhN-treated cells. Arrows indicate membrane associated Hhip (S). Myc-tagged Hhip (S, V), Clathrin (T, W) and merged image (U, X). (A–X) Single confocal images.
Reprinted from Developmental Biology, 297(1), Ochi, H., Pearson, B.J., Chuang, P.T., Hammerschmidt, M., and Westerfield, M., Hhip regulates zebrafish muscle development by both sequestering Hedgehog and modulating localization of Smoothened, 127-140, Copyright (2006) with permission from Elsevier. Full text @ Dev. Biol.