Journal: International Journal of Oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Depletion of uPAR and cathepsin B inhibits PKC/integrin signaling to FAK and the cytoskeletal molecules. A, Cell lysates from U251 and 5310 non-GICs and GICs were extracted using RIPA buffer after treatment with pUC and radiation alone or in combination, and western blot analysis was performed to determine the expression levels of FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, and α-actinin using their specific antibodies. GAPDH was used as a loading control. B, The total cell lysates of U251 and 5310 non-GICs and GICs as described above were immunoprecipitated with FAK antibody (2 μ g). The protein precipitates were washed with lysis buffer and incubated with 1X loading dye at 90°C for 10 min. SDS-PAGE was conducted and western blotting was performed with vinculin and α-actinin antibodies as described in Materials and methods.

Article Snippet: The following antibodies were used: uPAR, cathepsin B, CD133, CD44, Nestin, Sox-2, Ki67, Tuj-1, β-actin, PKC θ, integrin α2, integrin α5, integrin β1, FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, α-actinin, Rac-1, Cdc42, ERK, pERK, and GAPDH (all from SCBT, Santa Cruz, CA).

Techniques: Western Blot, Expressing, Control, Immunoprecipitation, Lysis, Incubation, SDS Page

Journal: International Journal of Oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: pUC inhibits the FAK migratory signaling pathway and inhibition of ERK can further influence the FAK signal. A, Cell lysates of control, pSV-transfected, and pUC-transfected U251 and 5310 non-GICs and GICs with and without irradiation were collected. Western blot analysis was performed for FAK migratory signaling molecules Rac-1, Cdc42, pCdc42/Rac-1, Ras, ERK, and pERK using their specific antibodies. GAPDH served as a loading control. B, The total protein lysates from U251 and 5310 non-GICs and GICs were collected from U0126 (10 μ M) and DMSO treatments. SDS-PAGE was conducted as described in Materials and methods. Western blotting was performed to determine the protein expression levels of ERK, pERK, FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, α-actinin, Rac-1, Cdc42, pCdc42/Rac-1, and Ras using their specific antibodies.

Article Snippet: The following antibodies were used: uPAR, cathepsin B, CD133, CD44, Nestin, Sox-2, Ki67, Tuj-1, β-actin, PKC θ, integrin α2, integrin α5, integrin β1, FAK, pFAK (Tyr 397 ), pFAK (Tyr 925 ), vinculin, α-actinin, Rac-1, Cdc42, ERK, pERK, and GAPDH (all from SCBT, Santa Cruz, CA).

Techniques: Inhibition, Control, Transfection, Irradiation, Western Blot, SDS Page, Expressing

Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 5. Depletion of uPAR and cathepsin B inhibits PKC/integrin signaling to FAK and the cytoskeletal molecules. A, Cell lysates from U251 and 5310 non-GICs and GICs were extracted using RIPA buffer after treatment with pUC and radiation alone or in combination, and western blot analysis was performed to determine the expression levels of FAK, pFAK (Tyr397), pFAK (Tyr925), vinculin, and α-actinin using their specific antibodies. GAPDH was used as a loading control. B, The total cell lysates of U251 and 5310 non-GICs and GICs as described above were immunoprecipitated with FAK antibody (2 µg). The protein precipitates were washed with lysis buffer and incubated with 1X loading dye at 90˚C for 10 min. SDS-PAGE was conducted and western blotting was performed with vinculin and α-actinin antibodies as described in Materials and methods.

Article Snippet: The following antibodies were used: uPAR, cathepsin B, CD133, CD44, Nestin, Sox-2, Ki67, Tuj-1, β-actin, PKC θ, integrin α2, integrin α5, integrin β1, FAK, pFAK (Tyr397), pFAK (Tyr925), vinculin, α-actinin, Rac-1, Cdc42, ERK, pERK, and GAPDH (all from SCBT, Santa Cruz, CA).

Techniques: Western Blot, Expressing, Control, Immunoprecipitation, Lysis, Incubation, SDS Page

Journal: International journal of oncology

Article Title: uPAR and cathepsin B knockdown inhibits radiation-induced PKC integrated integrin signaling to the cytoskeleton of glioma-initiating cells.

doi: 10.3892/ijo.2012.1496

Figure Lengend Snippet: Figure 6. pUC inhibits the FAK migratory signaling pathway and inhibition of ERK can further influence the FAK signal. A, Cell lysates of control, pSV- transfected, and pUC-transfected U251 and 5310 non-GICs and GICs with and without irradiation were collected. Western blot analysis was performed for FAK migratory signaling molecules Rac-1, Cdc42, pCdc42/Rac-1, Ras, ERK, and pERK using their specific antibodies. GAPDH served as a loading control. B, The total protein lysates from U251 and 5310 non-GICs and GICs were collected from U0126 (10 µM) and DMSO treatments. SDS-PAGE was conducted as described in Materials and methods. Western blotting was performed to determine the protein expression levels of ERK, pERK, FAK, pFAK (Tyr397), pFAK (Tyr925), vinculin, α-actinin, Rac-1, Cdc42, pCdc42/Rac-1, and Ras using their specific antibodies.

Article Snippet: The following antibodies were used: uPAR, cathepsin B, CD133, CD44, Nestin, Sox-2, Ki67, Tuj-1, β-actin, PKC θ, integrin α2, integrin α5, integrin β1, FAK, pFAK (Tyr397), pFAK (Tyr925), vinculin, α-actinin, Rac-1, Cdc42, ERK, pERK, and GAPDH (all from SCBT, Santa Cruz, CA).

Techniques: Inhibition, Control, Transfection, Irradiation, Western Blot, SDS Page, Expressing

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 1. An altered FN matrix activates a signaling pathway that involves FAK, JNK, and p53. A, Western blots of cell lysates showing that the VH FN fragment decreased FAK phosphorylation at Tyr397, increased JNK phosphorylation, and decreased total p53 levels in primary human fibroblasts compared with cells incubated in control medium (C) or with the control VH FN fragment for 1, 3, and 7 h. Actin served as a loading control. B, after immunoprecipitation (IP) of the samples shown in A with an antibody against pJNK, Western blotting with primary antibodies against p53 and pFAK (Tyr397) pulled down p53 and pFAK, respectively, consistent with complex formation between pJNK and p53 and between pJNK and pFAK. C, fluorescence images showing the localization of FAK, pJNK, and p53 in primary human fibroblasts treated with the VH protein or control medium (CTL) for 6 h. Cells in the top row were labeled first with primary antibodies against FAK, pJNK, and p53, then with secondary biotinylated antibodies, and finally with FITC-conjugated streptavidin. Cells in the bottom row were stained with DAPI nuclear stain. Original magnification, 400.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, Immunoprecipitation, Fluorescence, Labeling, Staining

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 2. FAK overexpression suppresses JNK phosphorylation and increases p53 levels, whereas dominant-negative con- structs FRNK and FAT and FAK antisense treatment increase JNK phosphorylation and decrease p53 levels under altered matrix conditions. The diagram at the top depicts the three FAK domains, the N-terminal, catalytic, and C-terminal domains, and two of the major phosphorylation sites (Y397 and Y925). It also illustrates that the C-terminal domain is known as the FRNK domain, which contains the FAT region. A, Western blots illustrate the levels of FAK, pFAK (phosphorylated at Tyr397 or Tyr925), pJNK, and p53 in primary human fibroblasts transfected with FAK, FRNK, FAT or a vector control and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated control. Actin served as a loading control. B, Western blots of cells were transfected with an antisense FAK oligonucleotide (AS FAK), a scram- bled FAK oligonucleotide control (Scr FAK), or a vector control (CTL) and treated as described in A.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Over Expression, Phospho-proteomics, Dominant Negative Mutation, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 3. P53 levels are suppressed by overexpression of JNK1 and in- creased by expression of a dominant- negative JNK1 construct. A, Western blots showing the levels of JNK1, pJNK, JNK2, FAK, pFAK (phosphorylated at Tyr397), and p53 in primary human fibro- blasts transfected with JNK1 or vector control or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated control. Actin served as a loading control. B, Western blots of pri- mary human fibroblasts transfected (Transf) with a dominant negative con- struct of JNK (JNK1 dn) or a vector con- trol or mock transfected and treated as described in A.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Over Expression, Expressing, Dominant Negative Mutation, Construct, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 4. Antisense JNK1 treatment increases p53 levels. A, West- ern blots showing the levels of JNK1, FAK, pFAK (phosphorylated at Tyr397), pJNK, and p53 in primary human fibroblasts cells transfected with an antisense JNK1 (AS JNK1) oligonucleotide or a scrambled JNK1 oligonucleotide (Scr JNK1) or mock transfected and treated with the VH fragment for 1, 3, and 7 h. C, untreated controls. Actin served as a loading control. B, Western blots showing JNK1, pJNK, and p53 levels in primary human fibroblasts transfected with a different anti- sense JNK1 oligonucleotide (AS JNK1*), a scrambled JNK1 oligonu- cleotide (Scr JNK1) or mock transfected and treated with the VH

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 5. Overexpression of JNK2 does not suppress p53 protein levels. Western blots showing the levels of JNK2, pJNK, JNK1, FAK, pFAK (phosphorylated at Tyr397), and p53 in primary human fibro- blasts transfected with JNK2 or vector control or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated controls. Actin was used as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 6. Antisense JNK2 treatment decreases p53 protein levels. A, Western blots showing the levels of JNK2, FAK, pFAK (phosphoryl- ated at Tyr397), pJNK, and p53 in primary human fibroblasts trans- fected with an antisense JNK2 oligonucleotide (AS JNK2) or a scram- bled JNK2 oligonucleotide (Scr JNK2) or mock transfected and treated with the VH FN fragment for 1, 3, and 7 h. C, untreated controls. Actin served as a loading control. B, Western blots showing JNK2, pJNK, and p53 levels in primary human fibroblasts transfected with a different antisense JNK2 oligonucleotide (AS JNK2*) or a scrambled JNK2 oligonucleotide (Scr JNK2) or mock transfected and treated with the VH fragment for 1, 3, and 7 h. Actin served as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Western Blot, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 7. P53 expression is suppressed in JNK2-deficient cells. A, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), pJNK, JNK2, and p53 in jnk2-deficient or control (CTL) cells treated with the VH FN fragment for 1, 3, or 7 h. C, untreated controls. Actin served as a loading control. B, Western blot showing p53 levels in jnk2-deficient cells from two different sources and in wild-type controls. Equal protein was loaded in each sample analyzed. C, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), JNK1, JNK2, and pJNK in mouse fibroblasts derived from jnk1/jnk2 double knock-outs or control cells treated with the VH fragment for 1, 3, and 7 h. Actin served as a loading control. D, Western blot showing p53 levels in mouse fibroblasts derived from jnk1/jnk2 double knock- outs and wild-type controls. Equal protein was loaded in each sample analyzed.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Expressing, Western Blot, Control, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: JNK1 and JNK2 Oppositely Regulate p53 in Signaling Linked to Apoptosis Triggered by an Altered Fibronectin Matrix

doi: 10.1074/jbc.m500331200

Figure Lengend Snippet: FIG. 8. P53 can feed back on pJNK to decrease pJNK protein levels in this pathway. Overexpressing p53 leads to a greater de- crease in pJNK levels, whereas knocking out the p53 gene leads to greater increases in pJNK protein levels in this pathway. A, Western blots showing the levels of p53, pJNK, JNK1, JNK2, FAK, and pFAK (phosphorylated at Tyr397) in primary human fibroblasts transfected with p53 or vector control or mock transfected and treated with the VH fragment for 1, 3, and 7 h. C, untreated controls. Actin was used as a loading control. B, Western blots showing the levels of FAK, pFAK (phosphorylated at Tyr397), pJNK, JNK2, JNK1, and p53 in p53 knock- out cells and wild-type controls treated with the VH FN fragment for 1, 3, and 7 h. Actin served as a loading control.

Article Snippet: The primary antibodies were rabbit anti-human JNK1 (C-17), mouse antihuman JNK2 (D-2), mouse anti-human pJNK (G-7), rabbit anti-human FAK (C-20), goat anti-human pFAK (Tyr 925), mouse anti-human p53 (DO-1), anti-mouse p53 (pab 240), and goat anti-human actin (I-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit polyclonal anti-FAK (pY397) phosphospecific antibody, rabbit polyclonal antiJNK1 and -2 SAPK (pTpY 183/1850) phosphospecific antibody (all from BIOSOURCE, Camarillo, CA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Knock-Out

Journal: FEBS Open Bio

Article Title: The molecular mechanism by which saturated lysophosphatidylcholine attenuates the metastatic capacity of melanoma cells

doi: 10.1002/2211-5463.12152

Figure Lengend Snippet: Lyso PC C18:0 affects focal adhesion complex formation in B16.F10 cells. (A) Western blotting images of FAK and the Tyr925 phosphorylated (p) FAK in B16.F10 cells as an indicator for focal adhesion complex formation dependent on pretreatment with Lyso PC . Prior to cell lysis, the cells were grown without fibronectin and treated with BSA 2%, BSA 2% plus Lyso PC C18:0 or Lyso PC C18:1 at concentrations of 450 μ m or only with DMEM for 72 h. Considering identical levels of FAK , the data indicate a clear reduction of FAK activation in the case of Lyso PC C18:0 pretreatment, whereas Lyso PC C18:1 apparently leaves the FAK activation unaffected. Representative blots from three independent experiments are shown. (B) Microscopic FAK phosphorylation analysis. Prior to application of antibodies, the cells were cultivated with DMEM for 72 h on fibronectin and treated with BSA 2% or, additionally, with 450 μ m Lyso PC C18:0 or Lyso PC C18:1. Cells were labeled with a fluorescent FAK antibody (large images). Identical areas at the leading edge of cells were selected to focus on activation in the migratory front and labeling of FAK (green) and pFAK (red) was compared. (C) A pixel density analysis was performed comparing the identical zoomed in cell areas at the cell lamellipodia in the FAK and pFAK labeled B16.F10 cells. The ratio of pFAK and FAK intensity is indicated and the lowest value for Lyso PC C18:0 pretreated cells is displayed, indicating the lowest FAK activation in line with the blotting data, whereas Lyso PC C18:1 obviously is without effect.

Article Snippet: Primary [FAK rabbit, pFAK (Tyr 925) goat] and secondary (rabbit FITC, goat CFL 647) antibodies were purchased from Santa Cruz Biotechnology Inc.

Techniques: Western Blot, Lysis, Activation Assay, Phospho-proteomics, Labeling