Figure 5.

Rumi cells can be transfected and are susceptible to RNAi. (A) The left panel shows a phase contrast image and the right panel shows a fluorescent image. One Rumi line-5 cell in the field is expressing MAPK-YFP. Cells were transfected with an Act5C-ERK-YFP plasmid⁸ using Effectene (Qiagen) and assayed for GFP expression 48 hours after transfection. (B) Western blot of Rumi-1 and Rumi-4 cells treated with either a control ds-GFP RNA or ds-arm RNA.⁷ The ds-arm RNA substantially reduces the level of Armadillo protein in cells from both cell lines. β-tubulin levels show similar amounts of proteins were loaded. PCR products corresponding to armadillio and GFP were used in an in vitro transcription reaction (Megascript, Ambion) to generate double stranded RNA. Rumi cells were seeded in 6-well plates 24 hrs prior to transfection with 30 μg dsRNA using Effectene (Qiagen). Cells were assayed after 48 hrs by Western blotting with monoclonal antibodies against Armadillo (1/100, N2 7A1, Developmental Studies Hybridoma Bank) and polyclonal antibodies against β-tubulin (1/2000 SC33749, Santa Cruz Biotechnology).