Skip to main content
. Author manuscript; available in PMC: 2015 Jul 23.
Published in final edited form as: Cell Rep. 2015 Jul 9;12(3):511–524. doi: 10.1016/j.celrep.2015.06.044

Figure 6. N-Ras controls IL8 induction by activating JAK2.

Figure 6

(A) The media harvested from control and N-RAS-overexpressing SUM102PT cells (n = 3 separate experiments) were analyzed for IL8 by ELISA. (B) The lysates of control or N-RAS-repressed cells (n=3 triplicates) were analyzed by ELISA for total and phosphorylated JAK2. (C) The control and N-RAS-repressed cells were examined by Western blot for indicated proteins. (D) The control and N-RAS-overexpressed cells, which were treated with or without TG101348, were seeded in soft agar for colony formation. (E) N-Ras was overexpressed as above, and the cells were treated with TG101348 to measure IC50. (F) N-RAS-silenced cells were also transduced to express JAK2-(V617F) before the growth rates (left) and colony formation in soft agar (middle) relative to the non-silenced control were measured. Protein expression levels were examined by Western blot (right). (G) HT1080 cells were transfected to express indicated Yn and Yc-tagged proteins and seeded into 96-well plates in triplicate (n = 3). The reconstituted YFP signal was detected by FACS (gated by untransfected cells as control), and an example of this is shown on the left. The percent YFP-positive cells was quantified and shown in the middle. Cells were also examined by Western blot to show that tagged N-Ras and JAK2 are expressed at comparable levels in various samples (right). (H) HT1080 cells carrying Yn and Yc-tagged proteins were examined by FACS. (I) HT1080 cells expressing Yn- and Yc-tagged proteins, as well as CFP-tagged cell compartment markers (GalT for Golgi, Rab5A for early endosomes, and Rab7A for late endosomes), were examined by confocal microscopy. (J) Cells overexpressing wild type or palmitoylation-deficient N-Ras were seeded in soft agar for colony formation (left). The right panel shows that both N-Ras proteins were expressed at comparable levels determined by Western blot. (K) MDA-MB-468 cells expressing indicated N-Ras proteins were treated with TG101348 for 16 hours, and then examined by Western blot. See also Figure S6.