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. 2015 Nov 24;6:8853. doi: 10.1038/ncomms9853

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(a) Western blot analysis of Akt and Etk/Bmx in cell lines. Cell lysates of the indicated cell lines were analysed by western blot analysis. Application of anti-Etk/Bmx or anti-Akt antibodies showed that endogenous Etk/Bmx was present only in CL1 cells, an aggressive prostate cancer cell line, or following ectopic overexpression in HEK 293T cells. In contrast, Akt was abundantly present in all the cell lines analysed. (b) PAR₁- and PAR₂-GST-C-tails associate with Etk/Bmx and Akt. Specific association of the Akt-PH domain was seen in cells that do not express Etk/Bmx (for example, HEK 293T and HCT116 cells). HEK293T cell lysates overexpressing etk/bmx plasmid concentrations (0.5–2 μg) showed no association with the Akt-PH domain. Application of HEK293T cells transfected to overexpress Etk/Bmx showed effective immobilization on both PAR₁ and PAR₂ C-tails. Application of HCT116 colon cancer cells that do not express Etk/Bmx showed a marked association with Akt. (c) PAR₂ binds GST-PH-Akt. Specific association of the Akt-PH domain was seen in cells that do not express Etk/Bmx (for example, HEK 293T and HCT116 cells). HEK293T cell lysates overexpressing etk/bmx plasmid concentrations (0.5–2 μg) showed no association with the Akt-PH domain. (d) Western blot analysis. Levels of T7-Etk/Bmx following increased etk/bmx plasmid transfection are shown. β-actin was used as a loading control. (e) PAR₂ binds GST-PH-Vav3. Specific association of the Vav₃-PH domain was seen in cells without Etk/Bmx expression (for example, HEK 293T and HCT116 cells). HEK293T cell lysates overexpressing etk/bmx plasmid concentrations (0.5–2 μg) showed no association with the Vav₃-PH domain. When indicated, detection of PAR₂ was carried out using either SAM 11ab (Santa Cruz Biotechnology) at 1:500 dilution. (f) Alignment PH-domain sequences of Etk/Bmx, Vav₃ and Akt. (d) PAR₂ binds GST-PH-Vav3. Multiple sequence alignment of PH-Etk/Bmx, PH-Vav₃ and PH-Akt was performed using T-coffee (available at http://www.ebi.ac.uk/Tools/msa/tcoffee).