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. 2015 Jul 20;210(2):347–361. doi: 10.1083/jcb.201501018

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© 2015 Prospéri et al.

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Figure 1. — Myo1b-Tail interacts with EphB2 receptors and is phosphorylated depending on EphB2 kinase activity. (A) Flag-EphB2, Flag-EphB2-KD, or EGFP-Myo1b immunoprecipitated from Hek293T cell lysate (Input) using anti-EphB2 or anti-GFP antibodies or normal IgG were analyzed by SDS-PAGE and immunoblotted (IB) with anti-EphB2, anti-GFP, and anti-phosphorylated tyrosine (anti-P-Tyr) antibodies. 70% of the pull-down was loaded to detect the coIPs, whereas 20% was loaded to detect the immunoprecipitations and EGFP-Myo1b tyrosine phosphorylation. The antibodies did not detect any material when the immunoprecipitations were performed with normal IgG and tyrosine phosphorylation of Flag-EphB2 that coIP with Myo1b was hardly detectable in these conditions. (B) The different EGFP-tagged Myo1b recombinant domains pulled down with GFP-Trap from cells lysates (Input) also expressing Flag-EphB2 were analyzed by SDS-PAGE and immunoblotted with anti-GFP, anti-EphB2, or anti-phosphorylated tyrosine antibodies. Cell lysates contained a similar amount of total proteins as detected by Ponceau S and EphB2 receptors as detected with anti-EphB2 antibodies. (C) EphB2 does not coIP with EGFP when coexpressed with Flag-EphB2 in Hek293T cells. (D) The amount of Flag-EphB2 pulled down with Myo1b recombinant domains was quantified and normalized to the amount of Myo1b recombinant domains expressed. Data are shown as the mean of three experiments. Error bars represent ± SEM. (E) EGFP-Myo1b that coIP with Flag-EphB2 or Flag-EphB2-KD and Flag-EphB2 or Flag-EphB2-KD that coIP with EGFP-Myo1b were quantified and normalized to the amount of EGFP-Myo1b and Flag-EphB2 or Flag-EphB2-KD expressed in lysates. EGFP-Myo1b that coIPs with Flag-EphB2-KD is expressed as a percentage of EGFP-Myo1b that coIPs with Flag-EphB2 and Flag-EphB2-KD that coIPs with EGFP-Myo1b is expressed as percentage of Flag-EphB2 that coIPs with EGFP-Myo1b. Data are shown as the mean of two experiments. Error bars represent ± SEM. (F) Phosphorylated EGFP-Myo1b that coIP with Flag-EphB2 or Flag-EphB2-KD was quantified and normalized to the amount of EGFP-Myo1b expressed in the lysates and expressed as a percentage of phosphorylated EGFP-Myo1b that coIP with Flag-EphB2. Data are shown as the mean of two experiments. Error bars represent ± SEM. (G) Phosphorylated EGFP-Myo1b domains in cell also expressing Flag-EphB2 were quantified and normalized to the amount of phosphorylated EGFP-Myo1b. Data are shown as the mean of three experiments. Error bars represent ± SEM. (H) EGFP-Myo1b or EGFP-Myo1b-Tail were pulled down by GFP-Trap beads from Hek293T cells expressing also Flag-EphB2 or Flag-EphB2-KD. Flag-EphB2 and Flag-EphB2-KD were expressed at similar levels as judged by immunoblotting of the cell lysates (Input) with anti-EphB2 antibodies. Similar amounts of EGFP-Myo1b or EGFP-Myo1b-Tail were immunoprecipitated from cells expressing Flag-EphB2 and Flag-EphB2-KD as judged by Ponceau S. 20% of Flag-EphB2 and Flag-EphB2-KD that coIP with EGFP-Myo1b or EGFP-Myo1b-Tail were analyzed by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies. (I) Phosphorylated EGFP-Myo1b and EGFP-Myo1b-Tail were quantified, normalized to the expression of EGFP-Myo1b or EGFP-Myo1b-Tail, and expressed as a percentage of phosphorylated EGFP-Myo1b. Data are shown as the mean of two experiments. Error bars represent ± SEM. (J) EphB2 or EGFP-Myo1b were immunoprecipitated from LS174T cell lysates (Input) expressing or not EGFP-Myo1b and treated or not with ephrinB1-Fc for 10 min with anti-EphB2 antibodies or GFP-Trap before being analyzed by SDS-PAGE and immunoblotting with anti-EphB2 or anti-Myo1b and anti-phosphotyrosine antibodies.