Skip to main content
. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Nat Chem Biol. 2013 Feb 17;9(4):247–249. doi: 10.1038/nchembio.1184

Figure 1. RU-SKI 43 inhibits Hhat.

Figure 1

a) RU-SKIs inhibit Shh palmitoylation in vitro. Membranes from cells transfected with wild-type Hhat or the inactive Hhat D339A mutant were preincubated with DMSO or 12.5μM RU-SKIs, C-1 or C-2, then incubated with ShhN (wild-type or C24A) and 125I-iodo-palmitoylCoA. 125I-iodo-palmitoyl incorporation was normalized to that of ShhN + DMSO; each bar is Mean±SD (n=2–4). b) RU-SKI 43 inhibition kinetics with purified Hhat; each point is Mean ±SD (n=2). c,d) RU-SKIs inhibit Shh protein palmitoylation in cells. COS-1 cells expressing HA-Hhat and Shh were treated with DMSO or RU-SKIs (c, 25 μM, 16 h; d, 0, 10 or 20 μM RU-SKI 43, 5 h) and labeled with 125I-iodo-palmitate. Anti-Shh immunoprecipitates were analyzed by SDS-PAGE and phosphorimaging (upper panel) or Western blotting (c, lower panel; d, middle panel). Total cell lysates (TCL) were analyzed by Western blotting. e) RU-SKI 43 exhibits specificity for Hhat. COS-1 cells expressing H-Ras N17, Fyn or c-Src, were treated with RU-SKI 43 and labeled with the indicated fatty acids. f) RU-SKI 43 does not inhibit Porcupine. Mouse L Wnt-3a cells were transfected with FLAG-Porcupine or empty vector (EV), treated with DMSO, 10 μM RU-SKI-43, or 100 nM Wnt C59, and labeled with 125I-iodo-pentadecanoate. Full, uncut gel images are presented in Supplementary Figures 9–12.