Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 5 Involvement of miR-1247-3p in the Gallic acid (GA)-inhibited integrin/FAK cascade in DLD-1 cells. Cells were treated with 90 μM GA for 24 h, lysed for total RNA extraction, and subjected to (A) miRNA expression profiling assay via microarray analysis or (B) quantitative analysis of the miRNAs via qPCR. miRNAs with up- and downregulation in response to GA were labeled with red and green, respectively. (C) Cells were transfected with control vector (Ctl-miR) or anti-miR-1247-3p (anti-miR), treated with GA for 24 h, and subjected to the assessment of integrins, FAK, and paxillin by Western blot. Densitometric analysis was performed for semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: RNA Extraction, Expressing, Microarray, Labeling, Transfection, Control, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 4 Gallic acid (GA) reduced integrin expression and inhibited FAK/Paxillin/Src and PI3K/AKT signaling in DLD-1 cells. Cells were treated with GA at the indicated concentrations for 24 h; collected; and lysed for the immunodetection of (A) integrins and the associated signaling proteins, (B) phosphor-paxillin and Src, and (C) PI3K, phosphor-AKT, and AKT via Western blot. Semi-quantitation of signals was conducted by densitometric analysis. DMSO treatment was used as control (C). Densitometric analysis was performed for the semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: Expressing, Immunodetection, Western Blot, Quantitation Assay, Control

Journal:

Article Title: A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

doi: 10.1091/mbc.E03-01-0046

Figure Lengend Snippet: Phosphorylation of FAK by α5β1 binding to substrate fibronectin in HT1080 cells. HT1080 cells were incubated in serum-free medium for 18 h, washed, dissociated with EDTA, and plated on fibronectin-coated, BSA-blocked, surfaces in complete PBS plus 2 mM glucose for 1 h at room temperature. The cells were extracted with a RIPA-vanadate buffer and analyzed by Western blot using antibodies to specific FAK phosphorylation sites or a mAb to FAK protein. (A) A representative blot; (B) the quantified combined data from three independent experiments FAK tyrosine phosphorylation (level of FAK detected by the specific antiphosphotyrosine antibody/level of FAK protein for each data point ×10). Error bars, SEM for n = 3 separate experiments. Linear regression: pY397 R2 = 0.98, p = 0.0009; pY407 R2 = 0.66, p = 0.09; pY577 R2 = 0.87, p = 0.02; pY861 R2 = 0.99, p = 0.0004. (C) The data as a function of the level of cross-linked α5. Linear regression: pY397 R2 = 0.99, p <0.0001; pY407 R2 = 0.72, p = 0.07; pY577 R2 = 0.84, p = 0.03; pY861 R2 = 0.97, p = 0.0017.

Article Snippet: Polyclonal anti-FAKpY397, FAKpY407, FAKpY577, FAKpY861, and FAKpY925 phosphospecific antibodies were obtained from Biosource (Camarillo, CA), polyclonal anti-FAK (A-17) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and monoclonal anti-FAK antibody was purchased from Transduction Laboratories (Lexington, KY).

Techniques: Phospho-proteomics, Binding Assay, Incubation, Western Blot

Journal:

Article Title: A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

doi: 10.1091/mbc.E03-01-0046

Figure Lengend Snippet: Phosphorylation of FAK by clustering integrin. HT1080 cells were incubated in serum-free medium for 18 h, washed, and dissociated with EDTA. Some samples were mixed with the mAb AIIB2, or AIIB2 plus secondary goat anti-rat IgG and plated on fibronectin-coated (335 ng/cm2), BSA-blocked surfaces in complete PBS plus 2 mM glucose for 10 and 60 min at room temperature. (A) A typical Western blot; (B) the quantification of Western blots for specific phosphorylated FAK; (phospho-specific antibody/total FAK) for each. Values were normalized to the phosphorylation level of the suspended cells in the absence of cluster inducing antibodies. Error bars, SEM for n = 3.

Article Snippet: Polyclonal anti-FAKpY397, FAKpY407, FAKpY577, FAKpY861, and FAKpY925 phosphospecific antibodies were obtained from Biosource (Camarillo, CA), polyclonal anti-FAK (A-17) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and monoclonal anti-FAK antibody was purchased from Transduction Laboratories (Lexington, KY).

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal:

Article Title: A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

doi: 10.1091/mbc.E03-01-0046

Figure Lengend Snippet: FAK stimulation by activated or ligand occupied α5β1. (A) Serum-starved HT1080 cells were treated is suspension with the β1 activating mAb AG89 or AG89 plus secondary anti-mouse IgG, or AG89 was bound to a nitrocellulose-coated surface. Cells kept in suspension or plated on fibronectin were used as controls. The specific phosphorylation is shown only for Y397 and Y861, there were no changes in Y407 or Y577. Data were normalized setting the specific phosphorylation for cells plated on fibronectin at 100%; error bars, SEM for n = 3. (B) Serum-starved HT1080 cells were treated in suspension with FN7–10 (40 μg/ml) and MnCl2 (1 mM) for 15 min; 13G12 (10 mg/ml) was added followed by secondary goat anti-mouse IgG (10 μg/ml) for 60 min. For controls, cells were maintained in suspension, plated on fibronectin, or plated on FN7–10 bound to 13G12 bound to nitrocellulose-coated surfaces. Quantification as for A, Error bars, SEM for n = 3.

Article Snippet: Polyclonal anti-FAKpY397, FAKpY407, FAKpY577, FAKpY861, and FAKpY925 phosphospecific antibodies were obtained from Biosource (Camarillo, CA), polyclonal anti-FAK (A-17) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and monoclonal anti-FAK antibody was purchased from Transduction Laboratories (Lexington, KY).

Techniques: Suspension, Phospho-proteomics