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. Author manuscript; available in PMC: 2015 Dec 20.
Published in final edited form as: FEBS Lett. 2014 Nov 20;588(24):4815–4824. doi: 10.1016/j.febslet.2014.11.016

Figure 2. spGFP:WNT1 (209-239) is palmitoylated in a PORCN-dependent manner.

Figure 2

HEK293T cells were co-transfected with WNT1 or spGFP:WNT1 (209-239) along with GFP (control) or PORCN. Cells expressing WNT1 (full length or the spGFP:Wnt1(209-239) fusion protein) were metabolically labeled with 3H-palmitate. Full length WNT1 was immunoprecipitated with anti-WNT1 antibodies while the fusion protein was immunoprecipitated with anti-GFP antibodies. Proteins were separated via SDS-PAGE, electroblotted onto a PVDF membrane, treated with En3Hance reagent, and subjected to autoradiography for 3 months. (A) PORCN promotes the incorporation of 3H-palmitate into full length WNT1 and spGFP:WNT1(209-239). 3H-palmitate is incorporated into monoglycosylated (upper band) and non-glycosylated (lower band) spGFP:WNT1(209-239). No 3H palmitate was incorporated into the S224A variant of the spGFP:WNT1 fusion protein. The Western blot of immunoprecipitated WNT1 and spGFP:WNT1 shows that the levels of these proteins are relatively constant in the presence and absence of co-transfected PORCN. (B) The amount of 3H-palmitate incorporated into total cell lysates was equivalent. Due to the lengthy exposures required, this experiment was carried out only twice.