Figure 2.
Figure 2. Myo1b-4YF is less phosphorylated than Myo1b but coIP with EphB2 and does not alter EphB2 delivery to the plasma membrane. (A) Representative tandem mass spectra (simultaneous fragmentation of neutral loss product and precursor) for identification of EGFP-Myo1b-Tail phosphorylation sites after its immunoprecipitation with GFP-Trap from Hek293T cells also expressing Flag-EphB2. Liquid chromatography tandem mass spectrometry is shown for EGFP-Myo1b-Tail peptides, with the position of the phosphate group monophosphorylated LIpY909EEKLEASELFKDK (679.34(3+) m/z), KALYPSSVGQPFQGApY938LEINKNPK (911.13(3+) m/z), LpY1049RTTLSQTK (645.83(2+) m/z), and diphosphorylated KALpY926PSSVGQPFQGApY938LEINKNPK (937.45(3+) m/z). The fragmentation spectra shown are Lys-C–derived peptides from EGFP-Myo1b-Tail. The corresponding peptide sequences and observed ions obtained from the phosphopeptides are shown above the spectra. Tandem mass spectrum are labeled to show singly, doubly, and triply charged b and y ions, as well as ions corresponding to neutral losses of phosphoric acid (P), water (circles), and NH3 (asterisks); M, parent ion mass. (B) EGFP-Myo1b and EGFP-Myo1b-4YF were pulled down with GFP-Trap from Hek293T cell lysates (Input) also expressing Flag-EphB2 and analyzed by SDS-PAGE and immunoblotting with anti-GFP, anti-EphB2, and anti-phospho-tyrosine (anti-P-Tyr) antibodies. Note the decrease of phosphorylation of EGFP-Myo1b-4YF compared with EGFP-Myo1b. (C) The amount of EphB2 that coIPs with EGFP-Myo1b or EGFP-Myo1b-4YF was quantified, normalized to the amount of the recombinant proteins pulled down, and expressed as a percentage of the amount that coIP with EGFP-Myo1b. Data are shown as the mean of three experiments. Error bars represent ± SEM. (D) The amount of phosphorylated EGFP-Myo1b and EGFP-Myo1b-4YF was quantified, normalized to the amount of the recombinant proteins pulled down, and expressed as a percentage of phosphorylated EGFP-Myo1b. Data are shown as the mean of three experiments. Error bars represent ± SEM. (E) YFP-EphB2-HCT cells transfected with Myo1b siRNAs and plasmid encoding Flag-HA-Myo1b-5M or Flag-HA-Myo1b-5M-4YF were incubated with clustered ephrinB1-Fc. The ratio of fluorescence detected at the cell surface for bound ephrinB1 over the fluorescence detected for YFP-EphB2 corresponding to the total amount of receptors was calculated for both experimental conditions and expressed in arbitrary units. Data are shown as the mean of two experiments (n = 84 for cells transfected with Myo1bsiRNA and Flag-HA-Myo1b-5M and n = 79 for cells transfected with Myo1bsiRNA and Flag-HA-Myo1b-5M-4YF). Note that the difference is not significant.

Myo1b-4YF is less phosphorylated than Myo1b but coIP with EphB2 and does not alter EphB2 delivery to the plasma membrane. (A) Representative tandem mass spectra (simultaneous fragmentation of neutral loss product and precursor) for identification of EGFP-Myo1b-Tail phosphorylation sites after its immunoprecipitation with GFP-Trap from Hek293T cells also expressing Flag-EphB2. Liquid chromatography tandem mass spectrometry is shown for EGFP-Myo1b-Tail peptides, with the position of the phosphate group monophosphorylated LIpY909EEKLEASELFKDK (679.34(3+) m/z), KALYPSSVGQPFQGApY938LEINKNPK (911.13(3+) m/z), LpY1049RTTLSQTK (645.83(2+) m/z), and diphosphorylated KALpY926PSSVGQPFQGApY938LEINKNPK (937.45(3+) m/z). The fragmentation spectra shown are Lys-C–derived peptides from EGFP-Myo1b-Tail. The corresponding peptide sequences and observed ions obtained from the phosphopeptides are shown above the spectra. Tandem mass spectrum are labeled to show singly, doubly, and triply charged b and y ions, as well as ions corresponding to neutral losses of phosphoric acid (P), water (circles), and NH3 (asterisks); M, parent ion mass. (B) EGFP-Myo1b and EGFP-Myo1b-4YF were pulled down with GFP-Trap from Hek293T cell lysates (Input) also expressing Flag-EphB2 and analyzed by SDS-PAGE and immunoblotting with anti-GFP, anti-EphB2, and anti-phospho-tyrosine (anti-P-Tyr) antibodies. Note the decrease of phosphorylation of EGFP-Myo1b-4YF compared with EGFP-Myo1b. (C) The amount of EphB2 that coIPs with EGFP-Myo1b or EGFP-Myo1b-4YF was quantified, normalized to the amount of the recombinant proteins pulled down, and expressed as a percentage of the amount that coIP with EGFP-Myo1b. Data are shown as the mean of three experiments. Error bars represent ± SEM. (D) The amount of phosphorylated EGFP-Myo1b and EGFP-Myo1b-4YF was quantified, normalized to the amount of the recombinant proteins pulled down, and expressed as a percentage of phosphorylated EGFP-Myo1b. Data are shown as the mean of three experiments. Error bars represent ± SEM. (E) YFP-EphB2-HCT cells transfected with Myo1b siRNAs and plasmid encoding Flag-HA-Myo1b-5M or Flag-HA-Myo1b-5M-4YF were incubated with clustered ephrinB1-Fc. The ratio of fluorescence detected at the cell surface for bound ephrinB1 over the fluorescence detected for YFP-EphB2 corresponding to the total amount of receptors was calculated for both experimental conditions and expressed in arbitrary units. Data are shown as the mean of two experiments (n = 84 for cells transfected with Myo1bsiRNA and Flag-HA-Myo1b-5M and n = 79 for cells transfected with Myo1bsiRNA and Flag-HA-Myo1b-5M-4YF). Note that the difference is not significant.

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