a b c d * MCF7.vec MCF7 IP: HA HA IgG p110 p  p85WT p85S83μ

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Presentation transcript:

a b c d * MCF7.vec MCF7 IP: HA HA IgG p110 p85 14-3-3 p85WT p85S83μ R X pS P K I S83 human bovine mouse rat S84 frog Mode 2 consensus S S231 L N231 C A N230 V Mode 1 consensus T Q S154 S153 IP: HA HA IgG p110 p85 14-3-3 p85WT p85S83μ p85S154/231μ p85 HA-14-3-3 IP: Xpress MCF7 c d Vector p85WT p85S83μ HA-14-3-3 p85 * MCF7 Supplementary Figure S3. 14-3-3 binds to serine 83 on p85 and activates PI3K/Akt. a. MCF7 HA-tagged 14-3-3ζ (MCF7) stable transfectants and their respective vector control cells (MCF7.vec) were immunoprecipitated (IP) with anti-HA or IgG followed by immunoblot with p110, p85, and 14-3-3 antibodies. b. Potential 14-3-3 binding motifs encompassing serine 83, serine 154 and serine 231 in human p85a compared to other organisms. c. MCF7ζ transfectants were stably transfected with empty vector (vector), histidine/Xpress-tagged (His/Xpress) wild type p85 (p85WT), or tagged p85 mutated in the 14-3-3 binding site encompassing S83 (p85S83μ) and immunoblotted with the indicated antibodies. Asterisk indicates tagged p85. d. MCF7 histidine/Xpress-tagged p85WT, p85S83μ or p85S154/231μ stable transfectants were immunoprecipitated (IP) with Xpress antibody followed by immunoblot with HA to detect exogenous 14-3-3ζ. Immunoprecipitation efficiency of Xpress-tagged p85 was determined by immunoblot with p85 antibody.