Figure S3.
Characterization of a P-Tyr280 JAM-A–specific antibody. (A) GST-fusion proteins containing the cytoplasmic domain of JAM-A, either the WT sequence (GST-hJAM-A/WT) or Tyr-to-Phe substitutions of the two Tyr residues present in the cytoplasmic domain of hJAM-A (-/Y261F, -/Y280F, -/Y261F_Y280F), were incubated with recombinant c-Src in kinase buffer. A fragment of the cytoplasmic domain of VE-cadherin (GST-VEcad/pm1) and GST alone (GST-) served as positive and negative controls, respectively. After kinase reaction, the GST fusion proteins were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel) or with a pan-P-Tyr-specific antibody (4G10 Platinum, middle panel). 10% of the input of GST fusion proteins were stained with Coomassie BB (bottom panel). Note that the P-Tyr280 JAM-A antibody detects JAM-A only when Tyr280 is unmodified. (B) Lysates of CHO cells ectopically expressing Flag-tagged JAM-A/WT (Flag-JAM-A/WT), Flag-JAM-A/Y280F, Flag-JAM-A/Y280E, or Flag-tagged JAM-C/WT (Flag-JAM-C/WT) were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel) or with a Flag tag-specific antibody (bottom panel). Note that the P-Tyr280 JAM-A antibody detects only WT JAM-A. (C) HEK293T cells were co-transfected with Flag-JAM-A constructs as indicated and constitutively active c-Src (Src/Y530F). Left panel: Flag immunoprecipitates were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel, 90% of input) or with a Flag tag-specific antibody (bottom panel, 10% of input). Right panel: Lysates of transfected cells were immunoblotted with an anti c-Src antibody (top) or with an anti-Flag antibody (bottom). Abbreviation: Trfct, Transfection; VEcad, VE-cadherin. Source data are available for this figure: SourceData FS3.

Characterization of a P-Tyr280 JAM-A–specific antibody. (A) GST-fusion proteins containing the cytoplasmic domain of JAM-A, either the WT sequence (GST-hJAM-A/WT) or Tyr-to-Phe substitutions of the two Tyr residues present in the cytoplasmic domain of hJAM-A (-/Y261F, -/Y280F, -/Y261F_Y280F), were incubated with recombinant c-Src in kinase buffer. A fragment of the cytoplasmic domain of VE-cadherin (GST-VEcad/pm1) and GST alone (GST-) served as positive and negative controls, respectively. After kinase reaction, the GST fusion proteins were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel) or with a pan-P-Tyr-specific antibody (4G10 Platinum, middle panel). 10% of the input of GST fusion proteins were stained with Coomassie BB (bottom panel). Note that the P-Tyr280 JAM-A antibody detects JAM-A only when Tyr280 is unmodified. (B) Lysates of CHO cells ectopically expressing Flag-tagged JAM-A/WT (Flag-JAM-A/WT), Flag-JAM-A/Y280F, Flag-JAM-A/Y280E, or Flag-tagged JAM-C/WT (Flag-JAM-C/WT) were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel) or with a Flag tag-specific antibody (bottom panel). Note that the P-Tyr280 JAM-A antibody detects only WT JAM-A. (C) HEK293T cells were co-transfected with Flag-JAM-A constructs as indicated and constitutively active c-Src (Src/Y530F). Left panel: Flag immunoprecipitates were separated by SDS-PAGE and immunoblotted with an anti P-Tyr280 JAM-A-specific antibody (Affi1550, top panel, 90% of input) or with a Flag tag-specific antibody (bottom panel, 10% of input). Right panel: Lysates of transfected cells were immunoblotted with an anti c-Src antibody (top) or with an anti-Flag antibody (bottom). Abbreviation: Trfct, Transfection; VEcad, VE-cadherin. Source data are available for this figure: SourceData FS3.

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