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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Int J Radiat Oncol Biol Phys. 2010 Jul 7;78(1):205–212. doi: 10.1016/j.ijrobp.2010.03.023

Figure 1. Knockdown of either RalA or RalB sensitizes MIA PaCa-2 pancreatic cancer cells to ionizing radiation (IR).

Figure 1

MIA PaCa-2 cells stably expressing shRNAs targeting RalA, RalB, K-Ras, or a non-specific sequence (NS) were generated. (a) Lysates were analyzed by immunoblotting (IB) to determine expression of RalA, RalB and K-Ras. β-actin was a loading control. Knockdowns of RalA, RalB and K-Ras were robust and specific. (b) Knockdown cells were analyzed by standard clonogenic growth assay following treatment with the indicated doses of IR. As expected, knockdown of K-Ras sensitized cells to IR. C Knockdown of either RalA or RalB also resulted in decreased survival post-IR. (c, d) shRNA-resistant (“rescue”) versions of either RalA or RalB were stably expressed in cells expressing RalA or RalB shRNAs, respectively. (c) Top, RalA protein re-expression was confirmed by IB; bottom, RalA re-expression rescued clonogenic growth defects associated with RalA shRNA expression. (d) Top, RalB protein re-expression was confirmed by IB; bottom, RalB re-expression rescued clonogenic growth defects associated with RalB shRNA expression. All results shown are representative of at least three independent experiments. Clonogenic survival assays were performed at least three times in triplicate. Bars represent mean ± standard deviation.