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. Author manuscript; available in PMC: 2009 Nov 16.
Published in final edited form as: Cancer Res. 2006 Aug 15;66(16):8200–8209. doi: 10.1158/0008-5472.CAN-06-0809

Figure 2.

Figure 2

SiRNA-mediated reduction of mutant V600EB-Raf expression reduces the downstream activity of Mek and Erk as well as levels of cyclin D1 protein in melanoma cells. A, siRNA-mediated knockdown of V600EB-Raf, but not of C-Raf, reduces phosphorylation (activity) levels of downstream proteins Mek and Erk in 1205 Lu cells 72 hours after nucleofection. Western blots of cells probed for the various proteins in the V600EB-Raf signaling cascade or control proteins following introduction of siRNA into cells. Erk 2 served as a control for protein loading. B, decreased cyclin D1 levels were detected in 1205 Lu cells following knockdown of V600EB-Raf expression. A 2-, 4-, 6-, and 8-day time course is shown for 1205 Lu cells following mutant V600EB-Raf inhibition. A decrease in cyclin D1 levels was observed starting at day 2 and ending at day 6. A corresponding increase in p27 levels was detected at day 2. Erk 2 was used as a protein loading control. SiRNA-mediated reduction of V600EB-Raf expression (activity) in melanoma cell lines UACC 903 (C) and A375M (D) led to significant decreases in activity and expression of downstream proteins 72 hours after nucleofection into cells. Increased p27 levels accompanied decreased cyclin D1 levels. Scrambled siRNA or buffer alone were used as controls. Erk 2 served as a control for protein loading.