pfak py397  (Cell Signaling Technology Inc)

Journal: BMC Cancer

Article Title: Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis

doi: 10.1186/1471-2407-11-375

Figure Lengend Snippet: Analysis of integrin surface expression (left) and intracellular integrin protein level (right) . PC-3 or LNCaP cells were treated either with 1 μM AEE788, 1 mM VPA or 1 nM RAD001, or with all compounds simultaneously (triple). Non-treated cells served as the controls. To explore integrin surface expression, cells were washed in blocking solution and stained with specific monoclonal antibodies as listed in materials and methods. A mouse IgG1-PE or IgG2a-PE was used as the isotype control. Fluorescence was analysed using a FACScan flow cytometer, and a histogram plot was generated to show PE-fluorescence. The mean fluorescence units are given in percentage difference to the controls. One of three independent experiments is shown here. *indicates significant difference to controls, #indicates significant difference to single drug treatment. To carry out western blotting, cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies (including anti-ILK, anti-FAK and anti-pFAK). β-actin served as the internal control. The figure shows one representative from three separate experiments.

Article Snippet: Additionally, integrin-related signaling was explored by anti-integrin-linked kinase (ILK; clone 3), anti-focal adhesion kinase (FAK; clone 77) and anti-phospho-specific FAK (pFAK; pY397; clone 18) antibodies (all: BD Biosciences).

Techniques: Expressing, Blocking Assay, Staining, Fluorescence, Flow Cytometry, Generated, Western Blot, SDS Page, Incubation

Journal: PLoS ONE

Article Title: Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

doi: 10.1371/journal.pone.0076232

Figure Lengend Snippet: (A) Expression of MMP-14 in B16F1 cells incubated 48h with 100µM lumcorin or scrambled peptide was analyzed by Western immunoblotting using polyclonal rabbit antibody and probing with anti-β-actin. (B, C) Activity of MMP-14 in B16F1 (B) and SK-MEL-28 (C) cells incubated 48h in the presence of 100µM lumcorin or its scrambled peptide, measured using fluorimetric SensoLyte® 520 MMP-14 Assay Kit as described in Materials and Methods. (D) Activity of MMP-14 in B16F1 cell extract pre-incubated 15 min or 60 min before assay with 100µM lumcorin or scrambled peptide. (E) Recombinant human MMP-14 activity pre-incubated 15 min with 100µM lumcorin or its scrambled peptide or DCN LRR9 or Fmod LRR9. Data are presented as mean ± S.D from three independent experiments. (F) Phospho-FAK (pY397) and total FAK expression in B16F1 cells incubated 15 min with 100µM lumcorin or scrambled peptide analyzed by Western immunoblotting using monoclonal mouse antibody against pFAK (pY397) and probing with polyclonal rabbit anti-total FAK antibody. After densitometric analysis of the intensity of the bands, the resulting ratio of pFAK to total FAK intensity was calculated and presented on the graph. Data are presented as mean values ± S.D from three independent experiments (*, p <0.05; **, p <0.01; ***, p <0.001; NS, no significant difference).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-human pFAK (pY397) (BD Biosciences, Bedford, MA, USA), rabbit polyclonal anti-mouse total FAK, rabbit polyclonal antibody directed against the hinge region of human MMP-14 (Abcam, Cambridge, UK), and goat anti-human actin (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Expressing, Incubation, Western Blot, Activity Assay, Recombinant