Journal: European journal of biochemistry

Article Title: Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator.

doi: 10.1046/j.1432-1033.2003.03549.x

Figure Lengend Snippet: Fig. 1. SDS/PAGE/Ligand blot analysis. The samples were reduced prior to electrophoresis. Left: SDS/PAGE indicates two bands of TN as a result of N-terminal cleavage. Right: Lanes 1–6 were loaded with plasminogen, tPA, uPA, HGF, prothrombin, and MSP, respectively. The blot shows TN-binding to plasminogen and HGF. However, longer exposure revealed binding to tPA as well.

Article Snippet: Ligand blot analysis using 125I-labelled tetranectin Three micrograms of bovine Plg, 4 lg human tPA, 2 lg human uPA, 2 lg human HGF (294-HGN, R&D Systems Europe, UK), 3 lg bovine prothrombin, and 2 lg human MSP (352-MS, R&D Systems) were dissolved in the Laemmli-buffer containing 2% SDS and subjected to SDS/PAGE.

Techniques: SDS Page, Electrophoresis, Binding Assay

Journal: eLife

Article Title: EGFR transactivates RON to drive oncogenic crosstalk

doi: 10.7554/eLife.63678

Figure Lengend Snippet: ( A and B ) HEK RON/EGFR or A431 RON cells were treated with ± 5 nM MSP or 50 nM EGF for 5 min at 37 °C. Representative immunoblots showing PY1068 and EGFR on cell lysates ( A ) or pan-phosphotyrosine (PY) and RON on samples immunoprecipitated (IP) with anti-HA antibody ( B ). ( C and D ) A431 RON cells were stimulated with ± 20 nM EGF, 20 nM MSP or both for 5 min at 37 °C and immunoblotted as in ( A and B ). Triplicate biological experiments are quantified in the bar graphs to the right, shown as mean ± SD. ( E ) Representative immunoblots of a phosphorylation time course for A431 RON cells treated with 5 nM EGF or 5 nM MSP and immunoblotted as in ( A and B ). Graphed values (right) are from triplicate biological experiments, normalized to maximal activation, and presented as mean ± SD. * p < 0.05; ** p < 0.01. Figure 1—source data 1. Full raw western blots and blots with relevant bands labeled, corresponding to . Figure 1—source data 2. Source data for quantification of blots in .

Article Snippet: Human recombinant EGF was from Invitrogen (cat # PHG0311) or PeproTech (cat # AF-100–15), biotin-conjugated and AF647-conjugated EGF from Thermo Fisher Scientific (cat # E3477 and E35351), and MSP from R&D Systems (cat # 4306 MS-010).

Techniques: Western Blot, Immunoprecipitation, Phospho-proteomics, Activation Assay, Labeling

Journal: eLife

Article Title: EGFR transactivates RON to drive oncogenic crosstalk

doi: 10.7554/eLife.63678

Figure Lengend Snippet: ( A and B ) A431 RON cells were pre-treated with 10 μM afatinib (Afat, pan-ErbB inhibitor) or 1 μM BMS777607 (BMS, Met family kinase inhibitor) for 20 or 15 min, respectively. Cells were then treated ± EGF or MSP for 5 min. ( A ) Cell lysates were used for PY1068 and EGFR immunoblots. ( B ) Lysates were immunoprecipitated (IP) with an anti-HA antibody and then immunoblotted for PY and RON. All samples are from the same blot, but an extraneous lane was removed for clarity. Bar graphs are corresponding mean ± SD from triplicate biological experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. Figure 5—source data 1. Full raw western blots and blots with relevant bands labelled, corresponding to . Figure 5—source data 2. Source data for quantification of blots in .

Article Snippet: Human recombinant EGF was from Invitrogen (cat # PHG0311) or PeproTech (cat # AF-100–15), biotin-conjugated and AF647-conjugated EGF from Thermo Fisher Scientific (cat # E3477 and E35351), and MSP from R&D Systems (cat # 4306 MS-010).

Techniques: Western Blot, Immunoprecipitation

Journal: eLife

Article Title: EGFR transactivates RON to drive oncogenic crosstalk

doi: 10.7554/eLife.63678

Figure Lengend Snippet: Cells were subsequently treated with EGF or MSP for 5 min and lysed for immunoblot analysis. For RON and phospho-RON immunoblotting, lysates were IP with an anti-HA antibody (RON) and whole cell lysates used for EGFR and EGFR-PY1068, and their respective overlays. Triplicate experiments for EGFR (white bars) and RON (grey bars) phosphorylation are plotted as mean ± SD. *** p < 0.001. Figure 5—figure supplement 1—source data 1. Full raw western blots and blots with relevant bands labelled, corresponding to . Figure 5—figure supplement 1—source data 2. Source data for quantification of blots in .

Article Snippet: Human recombinant EGF was from Invitrogen (cat # PHG0311) or PeproTech (cat # AF-100–15), biotin-conjugated and AF647-conjugated EGF from Thermo Fisher Scientific (cat # E3477 and E35351), and MSP from R&D Systems (cat # 4306 MS-010).

Techniques: Western Blot, Phospho-proteomics

Journal: eLife

Article Title: EGFR transactivates RON to drive oncogenic crosstalk

doi: 10.7554/eLife.63678

Figure Lengend Snippet:

Article Snippet: Human recombinant EGF was from Invitrogen (cat # PHG0311) or PeproTech (cat # AF-100–15), biotin-conjugated and AF647-conjugated EGF from Thermo Fisher Scientific (cat # E3477 and E35351), and MSP from R&D Systems (cat # 4306 MS-010).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Construct, Generated, Cytometry, Kinase Assay, Recombinant, Sequencing, Ligation, Mutagenesis, Bicinchoninic Acid Protein Assay, Expressing, Software, Magnetic Beads

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 1. RT-PCR analysis of Ron expression in various adult mouse tis- sues. The RNA from virgin uterus (lane 1), E12 placenta (lane 2), E16 placenta (lane 3), virgin cervix (lane 4), liver (5), colon (lane 6), epidid- ymis (lane 7), and testes (lane 8) were analyzed by RT-PCR using primers specific for mouse Ron (A), HGFL (B), or beta actin (C). The Ron amplified product was 404 bp and the HGFL amplified product 178 bp, whereas the beta actin product was 218 bp. As a negative control, water only was included in a PCR reaction (lane 9). A 100-bp ladder was included as a standard.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 4. RT-PCR analysis of Ron expression in murine trophoblast cell lines. The RNA from SM9-1 (lane 1), SM9-2 (lane 2), SM-10 (lane 3), and MEL (lane 4) cell lines were analyzed by RT-PCR using primers specific for Ron (A), HGFL (B), and beta actin (C) expression. The amplified Ron expression product was 404 bp. The amplified HGFL product was 178 bp, and the beta actin product was 218 bp. Water only was included as a negative control (lane 5). A 100-bp ladder was included as a standard.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 6. HGFL stimulates invasion of SM9-1 cells through Matrigel. In- vasion of SM9-1 cells through Matrigel-coated transwell membranes fol- lowing incubation for 24 and 48 h in F12 medium only (Serum Free), kallikrein-treated CHO conditioned medium (CHO/mock), or kallikrein- treated conditioned medium from CHO cells expressing HGFL (CHO/ HGFL) is shown. At 48 h, cellular invasion was significantly increased in the presence of HGFL compared to the mock-treated cells (*). Data shown are the average numbers of cells that had invaded through the Matrigel in 10 different visual fields with the standard deviation shown and P , 0.001. Experiments were performed in duplicate.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Incubation, Expressing, Standard Deviation

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 7. SM-10 cell survival following incubation in RPMI media con- taining 10% FBS, recombinant HGFL, or RPMI media only (Serum Free). The percentage of surviving cells was calculated for each time-point by counting the number of attached cells and dividing by the total number of cells counted (floating and attached). The absence of HGFL or 10% FBS produced a significant decrease in the percentage of surviving cells over 48 h (compare triangle- to square- and/or diamond-denoted lines). The experiment was performed in triplicate with P , 0.001 for those points indicated by the asterisks. To test whether HGFL could block SM-10 cells from undergoing apoptosis or necrosis, a TUNEL assay was per- formed (Fig. 8, A–D). SM-10 cells incubated for 24 h in the presence of serum or recombinant HGFL exhibited few- er TUNEL-positive cells compared to those cells incubated in serum-free medium (Fig. 8E).

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Incubation, Recombinant, Produced, Blocking Assay, TUNEL Assay

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 8. TUNEL staining of SM-10 cells following incubation in the presence or absence of HGFL. TUNEL analysis of fixed SM-10 cells following incubation for 24 h in RPMI with 10% FBS (B), RPMI with HGFL (C), or RPMI only (D) is shown. The negative (2) control (A) was incubated without terminal transferase. Percentage of TUNEL-positive cells per treatment group (A–D) is indicated in E. Percentages were calculated based on determining the num- ber of TUNEL-positive cells per four high- power areas, counting at least 100 cells per area. Standard error bars are indicated. *P , 0.003 compared to the negative con- trol in A, **P , 0.0001 compared to all other samples.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: TUNEL Assay, Staining, Incubation, Control