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. Author manuscript; available in PMC: 2016 May 9.
Published in final edited form as: Curr Biol. 2009 Dec 3;19(23):2020–2025. doi: 10.1016/j.cub.2009.10.040

Figure 2. RASSF1A Is Phosphorylated at Ser131 following Ionizing Radiation.

Figure 2.

(A) Alignment of a conserved SQ motif in RASSF1A sequences from vertebrates; the matching sequence in RASSF5 is included for comparison. Mutations that have been identified in human lung and ovarian cancer cells [8, 19] in proximity to Ser131 are highlighted in bold.

(B) MCF7 cells were transfected with pcDNA3 or plasmid expressing FLAG-RASSF1A or S131A-mutated FLAG-RASSF1A. Cells were harvested 30 min post γ-irradiation (10 Gy). FLAG-tagged proteins were immunoprecipitated and probed by western blot with the phosphospecific antibody raised against a phospho-Ser131 peptide spanning residues 125–136 of RASSF1A. Lysates and immunoprecipitates were blotted with the indicated antibodies. The data shown are representative of three independent experiments. Phospho-Ser131 bands were normalized to the loading controls, and bands were quantitated with ImageJ software. Error bars represent standard error.

(C) MCF7 cells were transfected with either pcDNA3 or plasmid expressing FLAG-RASSF1A or S131A-mutated FLAG-RASSF1A. Cells were irradiated (10 Gy) and harvested 30 min following irradiation. Endogenous MST2 was immunoprecipitated and probed by western blot for anti-MST2 and anti-FLAG. The kinase activity of MST2 in immunoprecipitates was determined by in gel kinase assay. Lysates were probed with indicated antibodies.