Journal: International journal of cancer

Article Title: Leptin promotes motility and invasiveness in human colon cancer cells by activating multiple signal-transduction pathways.

doi: 10.1002/ijc.23821

Figure Lengend Snippet: FIGURE 1 – Colon cancer cell lines express functional leptin receptor. LS174T cells grown to 90% confluence were lysed and western-blot analysis was performed for detection of the leptin receptor (a). Two bands were evident, of 100 and 125 kDa, corresponding to the short (Ob- Ra) and long (Ob-Rb) human isoforms, respectively. LS174T cells were serum-starved for 24 hr and treated with increasing doses of leptin (0, 10, 100 and 2,000 ng/ml) for 5 min (b, c), or with a single concentration of leptin (10 ng/ml) for increasing periods of time (0, 5, 10, 20, 30 and 40 min) (d, e). Cells were harvested and lysates were separated by SDS-PAGE and analyzed by immunoblotting. MAPK phosphorylation was detected with a specific phospho-MAPK antibody. The same blot was stripped and reprobed with an antibody against total ERK1/ERK2 (p44/ p42). For dose-dependent experiments, one representative of 2 independent experiments is shown with densitometric analysis in the lower panel representing the means of the experiments. For time-response experiments, 1 representative experiment from 3 independent ones is shown. HM7 cells were serum-starved for 24 hr and treated with increasing doses of leptin (0, 10, 100 and 2,000 ng/ml) for 5 min (e, f), or with a single con- centration of leptin (10 ng/ml) for increasing periods of time (0, 5, 10, 20, 30 and 40 min) (g, h). Cells were harvested and lysates were separated by SDS-PAGE and analyzed by immunoblotting. MAPK phosphorylation was detected with a specific phospho-MAPK antibody. The same blot was stripped and reprobed with an antibody against total ERK1/ERK2 (p44/p42). Densitometric analysis is shown and represent the means 6 SE of 3 independent experiments. Statistical difference was evaluated by one-way ANOVA. Means without a common letter differ, p < 0.05.

Article Snippet: The PhosphoPlus1 p44/42 MAP kinase (Thr202/Tyr204) antibody kit was from Cell Signaling Technology (Beverly, MA).

Techniques: Functional Assay, Western Blot, Concentration Assay, SDS Page, Phospho-proteomics

Journal: Oncogene

Article Title: Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells.

doi: 10.1038/sj.onc.1209592

Figure Lengend Snippet: Figure 4 Phosphotyrosine-binding domain (PTB) of SNT-1 (SNT-1 PTB) specifically reduces fibroblast growth factor (FGF)-1 stimulated mitogen-activated protein kinase (MAPK) phosphorylation when overexpressed in a doxycycline Dox regulatable manner. C9 cells stably transfected with pTREins/PTB or an empty vector were induced by Dox and stripped of estrogens as described in Figure 1b. Mitogen-activated protein kinase phosphorylation was induced by a 30 min incubation with FGF-1 (20 ng/ml) and heparin (50 mg/ml), or heregulin b1 (HRG-b1) (25 ng/ml), or 5% fetal borine serum. Total cellular protein (30 mg) was loaded in each well for immunoblotting with phospho-MAPK and MAPK antibodies.

Article Snippet: The PhosphoPlus AKT (Ser473) antibody kit, and the PhosphoPlus Map kinase (Thr202/Tyr204) antibody kit were from Cell Signaling Technology (Beverly, MA, USA) and the PhosphoPlus p70S6 kinase antibody kit (Thr389, Ser411, Thr421/Ser424) was from New England BioLabs (Beverly, MA, USA).

Techniques: Binding Assay, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Western Blot