DEV02881 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Porcupine promotes the lipid-modification of both wild-type Wnt-1/Wnt-3a and mutant Wnt-1/Wnt-3a, which possess C-to-A point mutations in the cysteine that is known to be palmitoylated. 293T cells were transfected with either Wnt1, Wnt3a, Wnt1(C93A) or Wnt3a(C77A) in the presence of either GFP (control) or mouse (m) PorcD. Cells were lysed 24 hours post-transfection and extracted with TX-114 before analysis by SDS-PAGE and western blot. The blots were probed with Wnt1 (left), Wnt3a (right) and tubulin antibodies. Monomeric Wnt1/Wnt3a proteins migrate at ∼40 kDa, whereas tubulin migrates at ∼50 kDa. In the absence of ectopic porcupine, Wnt1 and Wnt3a are found in both aqueous (A) and detergent (T) phases. The presence of ectopic porcupine promotes the partitioning of Wnt1, Wnt3a, Wnt1(C93A) and Wnt3a(C77A) into the hydrophobic phase.

• Supplemental Figure S2 -

  Fig. S2. Western blot analysis shows knockdown of human HA-PORC with PORC RNAi constructs. 293T cells were transfected with HA-tagged PORC alone or in combination with RNAi constructs targeting GFP or PORC (‘a’ and ‘b’ combined). After 24 hours, cells were lysed and subjected to SDS-PAGE followed by western blot analysis with anti-HA and anti-tubulin antibodies. After color development, the band intensities were quantitated using Image J software (NIH). The area under the peak for the HA-PORC band was divided by that for the tubulin band to account for any variation in loading. Because we used transient transfections for these assays, not all cells that were transfected with HA-PORC are expected to be transfected with the PORC RNAi constructs. In five out of five independent experiments, western blot analysis of cells transfected with HA-PORC and PORC RNAi show 21-45% less HA-PORC than cells transfected with HA-PORC and GFP RNAi.