antibody against phospho ron  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

doi: 10.1016/j.jbc.2022.102029

Figure Lengend Snippet: Comparison of cell migration versus proliferation in the presence of SSTN EGFR , EGFR kinase inhibitors, or kinase inhibitors specific for RON or ABL1. A , NOK or UM-SCC47 HNSCC cells were induced to migrate across LN332-coated filters in the presence or absence of 10 ng/ml EGF or 10 ng/ml EGF together with 30 μM SSTN EGFR , 3 μM gefitinib, 2 μM erlotinib, or 10 μg/ml α3β1 blocking antibody (P1B5) (Bar = 50 μM). Migration is quantified and expressed as percent of migration relative to EGF-stimulated cells; B , UM-SCC47 HNSCC or NOKs grown in complete culture medium were treated for 3 h with 30 μM SSTN EGFR , 3 μM gefitinib, or 2 μM erlotinib, then fixed and stained with EdU and DAPI to quantify the percentage of cells synthesizing new DNA relative to control cells treated with vehicle alone. Arrow highlights the decreased proliferation of UM-SCC47 cells in SSTN EGFR . Representative pictures show untreated versus treated UM-SCC47 cells, including a cohort treated with 30 μM SSTN EGFR supplemented with 10 ng/ml EGF (Bar = 50 μM); C , normal epithelial cells (NOKs, MCF10A), HNSCC cells (UM-SCC47, UM-SCC1), and TNBC cells (MB-468, MB-231) were grown for 3 h in complete culture medium containing RON kinase inhibitors (1 μM CAS 913376-84-8 or 3 μM BMS-77760l7) or ABL1 inhibitors (2 μM GNF5 or 0.2 μM PPY-A) followed by EdU labeling and quantification relative to control cells treated with vehicle alone; D , UM-SCC47 cells were treated for 3 h with vehicle or 30 μM SSTN EGFR , then lysed and the cell lysates were either (i) subjected to immunoprecipitation with nonspecific isotype control goat IgG or goat polyclonal anti-RON antibody (1 μg AF691/1 mg input) and analyzed on Western blots for total and active (pY1238/pY1239) RON and total and active (pY245 and pY412) ABL1 or (ii) analyzed on Western blots for active (pY245 or pY412) ABL1; ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001. EdU, 5-ethynyl-2′-deoxyuridine; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HNSCC, head and neck squamous cell carcinoma; LN332, laminin-332; NOK, normal oral keratinocytes; RON, recepteur d’origine nantais; TNBC, triple-negative breast cancer.

Article Snippet: Anti-Sdc4 (AF2918), EGFR (AF231), RON (AF691 and FAB6647F), pY1238/1239 RON (AF1947), ITGB4 (mAb 422325) Sdc2 (305515) were from R&D Systems .

Techniques: Comparison, Migration, Blocking Assay, Staining, Control, Labeling, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

doi: 10.1016/j.jbc.2022.102029

Figure Lengend Snippet: Identification of signaling receptors required for S-phase progression. A , UM-SCC47 cells treated with vehicle alone or 30 μM SSTN EGFR for 3 h were lysed and subjected to immunoprecipitation with nonspecific, isotype-matched control IgG, and mAb 8G3 to Sdc4 or rabbit polyclonal antibody to Sdc2. Immunoprecipitates were probed for the presence of EGFR, α3 integrin (ITGA3), β4 integrin (ITGB4), phosphorylated ITGB4 with PY20, CD151, total and active RON (pY1238/1239), total and active ABL1 (pY412 and pY245), and Sdc2 and Sdc4; B , GST-S4ED immobilized on glutathione beads was incubated with UM-SCC47 whole-cell lysates overnight in the presence or absence of 30 μM His-tagged S2ED and the receptors captured by S4ED were analyzed by Western blot; C , Sdc4 immunoprecipitates from HaCaT whole-cell lysates were probed for associated α3 integrin (ITGA3), β4 integrin (ITGB4), EGFR, RON, and Sdc2 via Western blot. RON and Sdc2 levels in the total lysate are shown. D , model showing proposed order of receptor complex assembly. E , UM-SCC47 cells transfected for 72 h with siRNA specific for human EGFR (3′UTR), α3 integrin (ITA3; s7543), CD151 (s194332), or β4 integrin (ITGB4; s7584) before performing Sdc4 immunoprecipitation and probing for associated receptors. Protein expression in starting cell lysates is shown in Fig. S2 A . Results are representative of duplicate experiments with cells transfected with two different siRNA oligos for each targeted protein (see Fig. S2 B ); F , UM-SCC47 cells were treated for 72 h with either control siRNA (AM4635) or siRNA-specific anti-human Sdc4 (12434), β4 integrin (ITGB4; s7584), α3 integrin (ITGA3; s7543), EGFR (3′ UTR), CD151 (s194332), Sdc2 (s12635), RON (s8996), or ABL1 (s865), then labeled with EdU to quantify DNA synthesis; ∗∗∗ p ≤ 0.001. Western blot inset shows individual receptor expression 72 h after siRNA transfection. Results are representative of duplicate experiments with cells transfected with two different siRNA oligos for each targeted protein. EGFR, epidermal growth factor receptor; RON, recepteur d’origine nantais; Sdc2, syndecan-2; Sdc4, syndecan-4.

Article Snippet: Anti-Sdc4 (AF2918), EGFR (AF231), RON (AF691 and FAB6647F), pY1238/1239 RON (AF1947), ITGB4 (mAb 422325) Sdc2 (305515) were from R&D Systems .

Techniques: Immunoprecipitation, Control, Incubation, Western Blot, Transfection, Expressing, Labeling, DNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

doi: 10.1016/j.jbc.2022.102029

Figure Lengend Snippet: Relative expression of members of the Sdc4:RTK:ITG complex on nontumorigenic and neoplastic epithelial cells. Cell surface of expression of Sdc4 (mAb 8G3, red ), Sdc2 (mAb 305515, orange ), α3β1 (mAb P1B5, blue ), α6β4 (mAb 3E1, green ), EGFR (mAb EGFR.1, black ), and RON (FAB6647F, purple ) were analyzed by flow cytometry compared to nonspecific IgG (gray profile) on A , HNSCC (UM-SCC47, UM-SCC1) and TNBC (MDA-MB-231) cells or B , nontransformed HaCaT, HTE, or NOK epithelial cells. EGFR, epidermal growth factor receptor; HNSCC, head and neck squamous cell carcinoma; HTE, human tonsillar epithelial cell; NOK, normal oral keratinocyte; RON, recepteur d’origine nantais; Sdc2, syndecan-2; Sdc4, syndecan-4; TNBC, triple-negative breast cancer.

Article Snippet: Anti-Sdc4 (AF2918), EGFR (AF231), RON (AF691 and FAB6647F), pY1238/1239 RON (AF1947), ITGB4 (mAb 422325) Sdc2 (305515) were from R&D Systems .

Techniques: Expressing, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Plasma membrane proteoglycans syndecan-2 and syndecan-4 engage with EGFR and RON kinase to sustain carcinoma cell cycle progression

doi: 10.1016/j.jbc.2022.102029

Figure Lengend Snippet: SSTN-induced cell cycle arrest depends on activated p38MAPK. A , quantification of p38MAPK activation by antibody array (pT180/pY182) in nontransformed keratinocytes (HTE, NOK, and HaCaT) and HNSCC cells (UM-SCC47, SCC25) treated with 30 mM SSTN EGFR ( left ) or in-cell western of UM-SCC47 cells following treatment with SSTNEGFR, RON kinase inhibitors (BMS-777607 or CAS-913376-84-8), or ABL1 inhibitors (GNF-5 or PPY-A)( right ); B , detection of pT180/pY182 p38MAPK following treatment with vehicle alone or 30 μM SSTN EGFR in nontransformed NOKs or transformed HNSCC cells; C , in nontransformed (MCF10A) or transformed (MDA-MB-231, MDA-MB-468, SKBr3) mammary epithelial cells; D , EdU-Click-IT–labeled UM-SCC47 cells treated for 3 h with either vehicle (control), 30 μM SSTN EGFR , 3 μM BMS-777607 (RON inhibitor), 0.2 μM PPY-A (ABL1 inhibitor), or 5 mM hydroxyurea (HU) in the presence or absence of 0.1 μM p38MAPK inhibitor BIRB-796 (Bar = 50 μm); E , quantification of EdU incorporation into cells treated with 30 μM SSTN EGFR with or without p38MAPK inhibitors BIRB-796 or Losmapimod for 3 h; ∗∗∗ p ≤ 0.001. EdU, 5-ethynyl-2′-deoxyuridine; HNSCC, head and neck squamous cell carcinoma; HTE, human tonsillar epithelial cell; NOK, normal oral keratinocyte; P38MAPK, p38 mitogen-activated protein kinase.

Article Snippet: Anti-Sdc4 (AF2918), EGFR (AF231), RON (AF691 and FAB6647F), pY1238/1239 RON (AF1947), ITGB4 (mAb 422325) Sdc2 (305515) were from R&D Systems .

Techniques: Activation Assay, Ab Array, In-Cell ELISA, Transformation Assay, Labeling, Control

Journal: Molecular Carcinogenesis

Article Title: A potential signaling axis between RON kinase receptor and hypoxia‐inducible factor‐1 alpha in pancreatic cancer

doi: 10.1002/mc.23339

Figure Lengend Snippet: RON and HIF‐1 α co‐expression in human PDAC tumors, pancreatic cancer cells, and invasive behavior: (A) Immunohistochemical analysis using RON and HIF‐1 α antibodies was performed on 101 PDAC tumors. RON and HIF‐1 α proteins are highly co‐expressed in all the tumors analyzed. H&E staining is shown to view the tissue histology. Scale bar 200 µm. (B) RON and HIF‐1 α protein expression was analyzed in a panel of pancreatic cancer cells with GAPDH as an internal control. HIF‐1 α protein expression under normoxia was observed in RON positive cells but not in RON negative cells. (C) RON and HIF‐1 α protein expression was analyzed in control and RON, HIF‐1 α knockdown or ectopic RON expressing pancreatic cancer cells. RON or HIF‐1 α knockdown significantly inhibited HIF‐1 α expression in RON positive cells while ectopic RON induced HIF‐1 α expression in RON null PDAC cells. GAPDH or actin is used as a loading control. (D) In vitro matrigel invasion assay either in the absence or presence of RON ligand, MSP (200 ng/ml) was performed in the control and RON, HIF‐1 α expression knockdown or RON expression rescued PDAC cells. MSP promoted the invasion of RON/HIF‐1 α positive control cells but significantly inhibited in the RON, HIF‐1 α expression knockdown cells, while RON expression rescued PDAC cells showed augmented invasion. (E) ImageJ software was used to measure the staining intensity of the invaded cells and quantification data representing the mean ± SD of three separate invasion assays was presented. * p < 0.05 and ** p < 0.01 [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Protein analysis was done on the total cell lysates using RON (SC‐74588), pRON (R&D AF1947), HIF‐1 α (CST‐14179), GAPDH (SC‐47724), Actin (SC‐4778), Caspase 3 (CST‐9662), Cleaved Caspase 3 (CST‐9661), pAKT (CST‐4060), and AKT (CST‐9272) antibodies.

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Knockdown, In Vitro, Invasion Assay, Positive Control, Software

Journal: Molecular Carcinogenesis

Article Title: A potential signaling axis between RON kinase receptor and hypoxia‐inducible factor‐1 alpha in pancreatic cancer

doi: 10.1002/mc.23339

Figure Lengend Snippet: Transcriptional regulation of HIF‐1 α through PI3K mediated RON Kinase pathway: (A) Western blot analysis on the total cell lysates from serum‐starved RON positive BXPC vector control, RON knockdown BXPC3 shRON cl2 cells and AKT inhibitor, MK‐2206 treated BXPC3 cells was performed with various antibodies. RON knockdown or AKT inhibitor, MK‐2206 inhibited PI3K mediated AKT phosphorylation and HIF‐1 α expression with no apparent changes in other proteins tested. (B) Quantitative real‐time RT‐PCR using RON, HIF‐1 α , and GAPDH internal control primers was performed on the total RNA from BXPC3 vector control, RON knockdown clones 2 and 4. RON knockdown significantly decreased HIF‐1 α mRNA expression. * p < 0.05. (C) Sp1 transcription factor effect on HIF‐1 α promoter activity was analyzed in breast cancer cells. An Sp dose‐dependent increase in HIF‐1 α promoter activity was observed. (D) HIF‐1 α promoter activity was analyzed in RON positive BXPC3 vector control in the absence or presence of Sp1 inhibitor, Mithramycin A, and RON knockdown BXPC3 sh RON cl2 cells. HIF‐1 α promoter activity was blunted in the Mithramycin A treated and RON knockdown cells

Article Snippet: Protein analysis was done on the total cell lysates using RON (SC‐74588), pRON (R&D AF1947), HIF‐1 α (CST‐14179), GAPDH (SC‐47724), Actin (SC‐4778), Caspase 3 (CST‐9662), Cleaved Caspase 3 (CST‐9661), pAKT (CST‐4060), and AKT (CST‐9272) antibodies.

Techniques: Western Blot, Plasmid Preparation, Control, Knockdown, Phospho-proteomics, Expressing, Quantitative RT-PCR, Clone Assay, Activity Assay

Journal: Molecular Carcinogenesis

Article Title: A potential signaling axis between RON kinase receptor and hypoxia‐inducible factor‐1 alpha in pancreatic cancer

doi: 10.1002/mc.23339

Figure Lengend Snippet: RON TKI, LCRF blocks MSP‐induced RON phosphorylation/activation and inhibits HIF‐1 α expression and invasion of pancreatic cancer cells. (A) Serum starved BXPC3 cells were stimulated with MSP 200 ng/ml either in the absence or presence of RON TKI, LCRF for the indicated time and phospho RON, total RON, and GAPDH protein expression was analyzed. MSP‐induced RON phosphorylation in the control cells, but RON TKI inhibited MSP‐induced RON phosphorylation. PDAC cells were grown in a serum‐free medium containing 200 ng/ml MSP for 24 h either in the absence or presence of RON TKI, LCRF, and HIF‐1 α , GAPDH protein expression was analyzed. MSP‐induced HIF‐1 α expression in the control cells, but RON TKI was effective at 400 nM concentration in significantly blocking the MSP‐induced HIF‐1 α expression. (B) Matrigel invasion assay was performed in the serum‐free medium containing 200 ng/ml MSP either in the absence or presence of RON TKI, LCRF. MSP‐induced invasion of both the pancreatic cancer cells. However, RON TKI at 400 nM concentration significantly inhibited MSP‐induced invasion. ImageJ software was used to measure the staining intensity of the invaded cells and quantification data representing three separate invasion assays was presented. * p < 0.05 and ** p < 0.01. (C) Pancreatic cancer cells were seeded in a 96‐well plate and incubated overnight. Cells are then changed to serum‐free medium containing 200 ng/ml MSP and grown for 24 h either in the absence or presence of RON TKI, LCRF. Cell proliferation was determined using nonradioactive CellTiter method. Very minimal alteration in the cell proliferation was observed in the control versus LCRF treated cells. (D) Pancreatic cancer cells were grown in the serum‐free medium containing 200 ng/ml MSP either in the absence or presence of RON TKI, LCRF. Total and cleaved Caspase 3 protein expression along with GAPDH control was analyzed. No apparent change in the total or cleaved Caspase 3 was observed between control and RON TKI treated pancreatic cancer cells [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Protein analysis was done on the total cell lysates using RON (SC‐74588), pRON (R&D AF1947), HIF‐1 α (CST‐14179), GAPDH (SC‐47724), Actin (SC‐4778), Caspase 3 (CST‐9662), Cleaved Caspase 3 (CST‐9661), pAKT (CST‐4060), and AKT (CST‐9272) antibodies.

Techniques: Phospho-proteomics, Activation Assay, Expressing, Control, Concentration Assay, Blocking Assay, Invasion Assay, Software, Staining, Incubation

Journal: Molecular Carcinogenesis

Article Title: A potential signaling axis between RON kinase receptor and hypoxia‐inducible factor‐1 alpha in pancreatic cancer

doi: 10.1002/mc.23339

Figure Lengend Snippet: RON knockdown inhibited HIF‐1 α expression in the human pancreatic tumor xenograft and RON, HIF‐1 α co‐expression in TNBC cells. (A) RON and HIF‐1 α expression was analyzed in the BXPC3 control and RON knockdown xenograft FFPE tissue. High RON and HIF‐1 α expression was detected in the BXPC3 control xenograft tissue. However, RON knockdown xenograft tissue exhibited a significantly decreased HIF‐1 α expression. Scale bar: 100 µm. (B) Immunofluorescence analysis was done on the invasive MDA MB 231, MDA MB 468 TNBC cells, and noninvasive MCF‐7 non‐TNBC cells. RON and HIF‐1 α co‐expression was detected in the TNBC cells but not in the non‐TNBC cells. (C) RON and HIF‐1 α protein expression was analyzed by Western blot analysis in the MDA MB 231 vector control and RON knockdown clones. RON and HIF‐1 α are expressed in the vector control cells, but RON knockdown significantly reduced HIF‐1 α expression [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Protein analysis was done on the total cell lysates using RON (SC‐74588), pRON (R&D AF1947), HIF‐1 α (CST‐14179), GAPDH (SC‐47724), Actin (SC‐4778), Caspase 3 (CST‐9662), Cleaved Caspase 3 (CST‐9661), pAKT (CST‐4060), and AKT (CST‐9272) antibodies.

Techniques: Knockdown, Expressing, Control, Immunofluorescence, Western Blot, Plasmid Preparation, Clone Assay

Journal: Cancers

Article Title: The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

doi: 10.3390/cancers12040904

Figure Lengend Snippet: RON is expressed in Ewing sarcomas and cell lines. ( a ) Relative RON transcript expression in Ewing sarcoma primary tumors from patients with localized (non-met) or metastatic (met) disease in comparison to MSC cultures, as determined by qPCR. ( b ) Respective RON expression in Ewing sarcoma cell lines (EwS) compared to MSC cultures. ( c ) RON protein is expressed and phosphorylated in Ewing sarcoma and rhabdomyosarcoma (RMS) cell lines. Cells were grown in standard tissue culture conditions. Following analysis of phospho-RON, blots were stripped and re-probed for total RON expression; 10% gel; numbers indicate densitometry readings relative to respective actin loading control.

Article Snippet: Primary antibodies detecting RON were Cat-No. HPA007657 (SEMA domain amino acids 283-433, corresponding to exons 1-2; unless otherwise specified, this antibody was used for total RON detection) and Cat-No. HPA008180 (IPT3 domain amino acids 767–875, corresponding to exons 9–10) from Sigma-Aldrich; phospho-RON (kinase domain Tyr1238/39) (Cat-No. AF1947) was from R&D Systems; MET (25H2) (Cat-No. 3127), phospho-MET (Tyr1234/35; D26) (Cat-No. 3077) and phospho-IGF1R (Tyr1131/1146) (Cat-No. 3021) were from Cell Signaling Technology (Beverly, MA, USA); IGF1Rβ (C20) (Cat-No. sc-713) and actin (C4) (Cat-No. sc-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Comparison, Control

Journal: Cancers

Article Title: The Receptor Tyrosine Kinase RON and Its Isoforms as Therapeutic Targets in Ewing Sarcoma

doi: 10.3390/cancers12040904

Figure Lengend Snippet: Ewing sarcomas express targeting-relevant RON isoforms. ( a ) Western blots suggest the presence of full-length RON (flRON) splice variants in pediatric sarcoma cell lines. RON protein expression was analyzed in comparison to characterized isoforms in HT-29 and HCT-116. Cells were grown in standard tissue culture conditions. Following analysis of phospho-RON, Western blots were stripped and re-probed for analysis of two distinct total RON antibodies directed at the SEMA and IPT3 domain epitopes. Arrows indicate RON species. 8% gel. ( b ) Ewing sarcomas express the short-form RON ( sfRON ) isoform containing (upper band) and/or lacking intron 11 sequences (lower band). Tumor samples are numbered; M indicates primary tumors from patients with metastatic disease, R indicates a relapsed tumor. ( c ) Sarcoma cell lines express sfRON . RT-PCR was performed on mRNA isolated from cell lines grown in standard conditions. ( d , e ) Treatment with 5-Aza-2’-deoxycytidine (5-Aza-CdR) modulates flRON ( d ) and sfRON ( e ) transcription. RT-PCRs were performed on mRNA isolated from cell lines grown in standard conditions and treated with 2.5 µM 5-Aza-CdR for 72 h where indicated. In ( a – e ), numbers indicate densitometry readings relative to the respective actin or GAPDH loading control.

Article Snippet: Primary antibodies detecting RON were Cat-No. HPA007657 (SEMA domain amino acids 283-433, corresponding to exons 1-2; unless otherwise specified, this antibody was used for total RON detection) and Cat-No. HPA008180 (IPT3 domain amino acids 767–875, corresponding to exons 9–10) from Sigma-Aldrich; phospho-RON (kinase domain Tyr1238/39) (Cat-No. AF1947) was from R&D Systems; MET (25H2) (Cat-No. 3127), phospho-MET (Tyr1234/35; D26) (Cat-No. 3077) and phospho-IGF1R (Tyr1131/1146) (Cat-No. 3021) were from Cell Signaling Technology (Beverly, MA, USA); IGF1Rβ (C20) (Cat-No. sc-713) and actin (C4) (Cat-No. sc-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Comparison, Reverse Transcription Polymerase Chain Reaction, Isolation, Control