Journal: PeerJ

Article Title: Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

doi: 10.7717/peerj.4063

Figure Lengend Snippet: In normal fibroblasts, well-developed stress fibers ((A) rhodamine-labeled phalloidin staining, (C) anti alpha-actinin staining) were observed in the center of the cells. Focal adhesions were observed in both the central and peripheral part of the cell ((E) anti-paxillin staining, (G) anti-FAK staining). In FAK −∕− , the stress fibers were seen around the cell, but the number of stress fibers in the center was small ((B) rhodamine-labeled phalloidin staining, (D) anti-alpha-actinin staining). The focal adhesions were well developed in the peripheral part, but the number of focal adhesions in the central part was small ((F) anti-paxillin staining). No staining was detected in FAK −∕− cells stained with anti-FAK antibody ((H) anti-FAK staining). Conventional epifluorescence microscopy. Bar; 20 µm

Article Snippet: The cells were then treated with 10% normal goat serum for 1 h at room temperature, and stained with monoclonal anti-FAK (1:100) (Takara, Tokyo, Japan), monoclonal anti-alpha-actinin (Sigma), anti-paxillin (BD Biosciences), polyclonal anti-tyrosine-phosphorylated FAK (pY-418; BioSource) antibody, or polyclonal anti-tyrosine-phosphorylated Src (pY-418; BioSource) for 60 min. After washing in PBS for 20 min, the fixed specimens were incubated with fluorescein-conjugated goat anti-rabbit or anti-mouse IgG.

Techniques: Labeling, Staining, Epifluorescence Microscopy

Journal: PeerJ

Article Title: Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

doi: 10.7717/peerj.4063

Figure Lengend Snippet: Summary of the number (A) and the area (B) of focal adhesions. Average of the number (A) and the area (B) of focal adhesions were shown. Control fibroblasts were normal fibroblast stained with anti-paxillin antibody. FAK −∕− +WT FAK gene was FAK −∕− cells transfected with wildtype RFP- FAK gene. Fibroblast + FRNK gene was fibroblasts transfected with GFP- FRNK gene. FAK −∕− cells were untreated FAK −∕− cells. Number of FAs at peripheral (or center) was an averaged number of focal adhesions located at the cell peripheral (or center). Area of FAs at peripheral (or center) was averaged area of focal adhesions located at the cell periphery (or center). When GFP-FRNK was introduced into FAK −∕− cells, the quantitative analysis was excluded, because there were no significant changes in the cell morphology or in the formation of stress fibers and focal adhesions. ± represents standard error of the mean.

Article Snippet: The cells were then treated with 10% normal goat serum for 1 h at room temperature, and stained with monoclonal anti-FAK (1:100) (Takara, Tokyo, Japan), monoclonal anti-alpha-actinin (Sigma), anti-paxillin (BD Biosciences), polyclonal anti-tyrosine-phosphorylated FAK (pY-418; BioSource) antibody, or polyclonal anti-tyrosine-phosphorylated Src (pY-418; BioSource) for 60 min. After washing in PBS for 20 min, the fixed specimens were incubated with fluorescein-conjugated goat anti-rabbit or anti-mouse IgG.

Techniques: Staining, Transfection

Journal: PeerJ

Article Title: Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts

doi: 10.7717/peerj.4063

Figure Lengend Snippet: Average of focal adhesion numbers (A) and areas were shown (B). For each experiment 10 cells were analyzed. In (B), the data were presented as an average ± standard error. Focal adhesion numbers and area were measured using Fiji software. Error bars represent standard error of the mean. P -value < 0.01. Cont PR, focal adehsion located at the cell periphery in normal fibroblast stained with anti-paxillin. Cont CT, focal adehsions located at the cell center in normal fibroblast stained with anti-paxillin. FAK −∕− WT PR, focal adhesions located at the cell periphery in FAK −∕− cells transfected with wild type RFP-FAK gene. FAK −∕− WT CT, focal adhesions located at the cell center in FAK −∕− cells transfected with wild type RFP- FAK gene. FRNK PR, focal adhesions located at the cell periphery in fibroblast transfected with GFP- FRNK gene. FRNK CT, focal adhesions located at the cell center in fibroblast transfected with GFP- FRNK gene. FAK −∕− PR, focal adhesions at the cell periphery in FAK −∕− cells. FAK −∕− CT, focal adhesions at the cell center in FAK −∕− cells.

Article Snippet: The cells were then treated with 10% normal goat serum for 1 h at room temperature, and stained with monoclonal anti-FAK (1:100) (Takara, Tokyo, Japan), monoclonal anti-alpha-actinin (Sigma), anti-paxillin (BD Biosciences), polyclonal anti-tyrosine-phosphorylated FAK (pY-418; BioSource) antibody, or polyclonal anti-tyrosine-phosphorylated Src (pY-418; BioSource) for 60 min. After washing in PBS for 20 min, the fixed specimens were incubated with fluorescein-conjugated goat anti-rabbit or anti-mouse IgG.

Techniques: Software, Staining, Transfection

Journal: Cancer Science

Article Title: B3 GNT 3 expression suppresses cell migration and invasion and predicts favorable outcomes in neuroblastoma

doi: 10.1111/cas.12294

Figure Lengend Snippet: Signaling molecules affected by B3GNT3 expression. (a) B3GNT3 decreases tyrosine phosphorylation (indicated by lowercase ‘p’) of FAK, Src and paxillin in SK‐N‐SH cells. The band intensity of pY118‐paxillin was normalized to the total protein. †The band intensity of pY397‐FAK and pY418‐Src was normalized to the ratio of their total protein to GAPDH, respectively. The results are representative of three independent experiments. The signals were quantified using ImageQuant 5.1 (Amersham Biosciences, Piscataway, NJ, USA). (b) B3GNT3 suppresses phosphorylation of Akt and ERK1/2 in SK‐N‐SH cells. The results are representative of three independent experiments. The signals were quantified using ImageQuant 5.1 (Amersham Biosciences) and were normalized to their controls.

Article Snippet: For cell signaling analyses, we used rabbit anti‐FAK pY397 polyclonal antibody (Biosource, Nivelles, Belgium), rabbit anti‐FAK polyclonal antibody (C‐20; Santa Cruz Biotechnology), anti‐paxillin pY118 (BD Transduction Laboratories, Heidelberg, Germany), anti‐paxillin polyclonal antibody (BD Transduction Laboratories), rabbit anti‐pERK1/2 monoclonal antibody, rabbit anti‐pAkt and mouse anti‐pan Akt monoclonal antibody (Cell Signaling Technology Inc., Beverly, MA, USA), rabbit anti‐ERK1/2 antibody (Santa Cruz Biotechnology), rabbit anti‐Src pY418 antibody (Invitrogen) and rabbit anti‐c‐Src antibody (Invitrogen).

Techniques: Expressing