epha2 protein cf  (R&D Systems)

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: A SRC–EPHA2–PI3Kβ tripartite complex drives oncogenic signaling in PTEN-null tumors. A, A schematic of the experiment for the targeted compound library screening to identify PI3Kβ phosphorylation inhibitors. Endogenous PI3Kβ-depleted BT549 or PC3 cells replaced with PI3Kβ-WT or PI3Kβ-Y962F mutants were treated with compounds from the library at 0.1 μmol/L. Cell viability was assessed by CCK-8 assays at 48 hours. B and C, Results of the kinase inhibitor screening in BT549 ( B ) and PC3 ( C ) cells. SRC inhibitors dasatinib and KX2-391 were among the top three significant inhibitors in both cells. D and E, Dasatinib effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 and PC3 cells. F, KX2-391 effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 cells. G, Dasatinib and KX2-391 dramatically reduced the growth of PPB cells with addback of PI3Kβ-WT but was not as effective on PPB cells with addback of PI3Kβ-Y956F. H, Knockdown of SRC or EPHA2 decreased phosphorylation of PI3Kβ-Y962 and c-MYC in BT549 cells. I, SRC coordinated with EPHA2 to phosphorylate PI3Kβ. SRC-Flag and EPHA2 plasmids were overexpressed in PTEN-WT and PTEN-KO HEK293T cells, and Flag was immunoprecipitated, followed by Western blot analysis. J, SRC and EPHA2 phosphorylated PI3Kβ-Y962 peptides in vitro . GST-SRC or GST-EPHA2 was incubated with synthetic Y962 containing PI3Kβ peptides in phosphorylation buffer, and the resulting peptides were analyzed by MS spectrum. K, In vitro phosphorylation assays showed that SRC and EPHA2 directly phosphorylated PI3Kβ-Y962, and the phosphorylation activity was blunted on PI3Kβ mutant at Y962. Particularly, SRC could strongly phosphorylate PI3Kβ-Y962. An in vitro kinase assay was performed by mixing GST-SRC or GST-EPHA2 with Flag-PI3Kβ-WT or Flag-PI3Kβ-Y962F proteins in the presence of ATP. Anti–phospho-PI3Kβ-Y962 antibody was used to detect the phosphorylated PI3Kβ-Y962. L, Knockdown of SRC (shSRC) dramatically decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; this effect can be slightly rescued by exogenous expression of EPHA2. M, Knockdown of EPHA2 (shEPHA2) decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; exogenous expression of SRC was unable to rescue the effect by sh EPHA2 in BT549 cells. N, SRC expression elevated p-ERK and c-Myc levels in BT549 cells, which were abolished by EPHA2 KO. O, A mechanism model to show that SRC collaborates with EPHA2 to phosphorylate PI3Kβ-Y962, whereas PTEN loss abolished PI3Kβ dephosphorylation, leading to PI3Kβ hyperphosphorylation and subsequent SRC–EPHA2–p-PI3Kβ Y962 complex formation to upregulate p-ERK/c-Myc signaling, accompanying with enhanced accessibility of PI3Kβ to phosphorylate PIP2 on the cell membrane to upregulate pAKT. Values were presented as the mean ± SEM. P values were determined by unpaired two-tailed t test (B, C). **** P < 0.0001.

Article Snippet: For peptide reactions, a 30 μL reaction system containing kinase reaction buffer added with 100 μmol/L ATP, 1 mmol/L DTT 250 ng of purified GST–SRC or GST-EPHA2 (MCE), and 0.05 mg/mL synthetic nonphosphorylated peptide were prepared.

Techniques: Drug discovery, Phospho-proteomics, CCK-8 Assay, Knockdown, Immunoprecipitation, Western Blot, In Vitro, Incubation, Activity Assay, Mutagenesis, Kinase Assay, Expressing, De-Phosphorylation Assay, Membrane, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Monoclonal neutralizing antibodies elicited by infection with Kaposi sarcoma-associated herpesvirus reveal critical sites of vulnerability on gH/gL

doi: 10.1371/journal.ppat.1013772

Figure Lengend Snippet: (A) Plasmids encoding the gH ectodomain and gL were co-transfected into 293 cells and recombinant gH/gL was purified from the supernatant. Made with BioRender.com. McGuire, A. (2025). https://BioRender.com/ks0xtn4 . (B) Recombinant gH/gL was subjected to size exclusion chromatography on a BioRad ENrichSEC 650 10 x 300 column. Three fractions were collected as indicated. (C) An aliquot of the gH/gL preparation pre-SEC, as well as equal amounts of the three fractions collected from B were analyzed by reducing SDS-PAGE followed by Coomassie staining. (D) Coomassie stained reducing SDS-PAGE gel of recombinant gH/gL treated, or untreated with PNGaseF as indicated. (E) Binding of recombinant gH/gL or recombinant Epstein-Barr virus gp350 to recombinant ephrin receptor A2 (EphA2) was measured by biolayer interferometry.

Article Snippet: Biotinylated EphA2 (Sino Biological Cat: 13926-H27H-B) was diluted to 10 μg/ml and immobilized on streptavidin biosensors for 300s, and then immersed in KB buffer for 60 seconds.

Techniques: Transfection, Recombinant, Purification, Size-exclusion Chromatography, SDS Page, Staining, Binding Assay, Virus

Journal: PLOS Pathogens

Article Title: Monoclonal neutralizing antibodies elicited by infection with Kaposi sarcoma-associated herpesvirus reveal critical sites of vulnerability on gH/gL

doi: 10.1371/journal.ppat.1013772

Figure Lengend Snippet: (A) gH/gL was incubated with, or without the indicated mAbs and the binding to immobilized EphA2 was measured by biolayer interferometry. The dashed line represents 100% binding set to the gH/gL alone control. (B-C) MLKH1/gH/gL (B) and MLKH5/gH/gL (C) complexes were purified and visualized by negative stain EM (nsEM). A representative 2D class average (top) and 3D reconstruction (bottom) are shown for each complex. The ribbon structure of gH/gL (PDB entry 7CZF, gH in black, gL in yellow) with each Fab (modeled using Alphafold) was fitted into the nsEM maps. (D) The crystal structure of the EphA2/gH/gL complex (PDB entry 7CZE) is shown for comparison.

Article Snippet: Biotinylated EphA2 (Sino Biological Cat: 13926-H27H-B) was diluted to 10 μg/ml and immobilized on streptavidin biosensors for 300s, and then immersed in KB buffer for 60 seconds.

Techniques: Incubation, Binding Assay, Control, Purification, Staining, Comparison