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