Journal: Journal of cell science

Article Title: Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

doi: 10.1242/jcs.01271

Figure Lengend Snippet: Fig. 3. Localization of F-actin in wild-type and transgenic corneas during wound healing in organ culture. Debridement wounds were made in corneas of wild-type and ALDH3-Cdk5 transgenic mice. Eyes were placed in organ culture for 12 hours and then fixed and stained with rhodamine-phalloidin. Corneas were dissected and whole mounted for laser scanning confocal fluorescence microscopy. (A) Rhodamine-phalloidin staining of the wound edge in a wild-type cornea. Open arrowheads indicate rows of cells with diffuse actin staining oriented perpendicular to the wound edge. Elongated cells are indicated by solid arrowheads. Points of intense actin staining are often located at the junctions of three or more cells (arrow). (B) Rhodamine-phalloidin staining of the wound edge in an ALDH3- Cdk5 cornea shows cobblestone organization along the wound edge, with few, if any, elongated cells. Cortical actin staining is distributed uniformly around the cell periphery and very few cells show diffuse staining. Bar, 100 µm.

Article Snippet: 25-50 μg of total cell extract were immunoblotted as previously described (Gao et al., 1997) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416cSrc (Cell Signaling Technology, Beverly, MA).

Techniques: Transgenic Assay, Organ Culture, Staining, Microscopy

Journal: Journal of cell science

Article Title: Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

doi: 10.1242/jcs.01271

Figure Lengend Snippet: Fig. 4. Effect of olomoucine on F-actin and Src activation at the wound edge. Debridement wounds were made in corneas of wild-type mice, eyes were organ cultured for 12 hours, fixed and double-stained with rhodamine-phalloidin (F-actin) and antibody to activated Src (Src pY416). Stained tissues were dissected, whole mounted and examined by confocal microscopy. (A) F-Actin staining in the absence of olomoucine shows a sharp, regular wound edge, with actin staining at cell-cell boundaries and along the wound edge. (B) In the presence of olomoucine, the wound edge is disorganized and cells have separated from the epithelial cell sheet (arrows). F-Actin staining is seen in a broader band of cells along the wound edge than in untreated corneas. (C) Immunostaining of active Src (Src pY416) shows that Src is activated in a narrow band of cells adjacent to the wound edge. (D) In the presence of olomoucine, immunostaining of active Src suggests that inhibiting Cdk5 augments the activation of Src in many cells along the wound edge (arrows). Bar, 125 µm.

Article Snippet: 25-50 μg of total cell extract were immunoblotted as previously described (Gao et al., 1997) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416cSrc (Cell Signaling Technology, Beverly, MA).

Techniques: Activation Assay, Cell Culture, Staining, Confocal Microscopy, Immunostaining

Journal: Journal of cell science

Article Title: Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

doi: 10.1242/jcs.01271

Figure Lengend Snippet: Fig. 6. Effect of Cdk5 and Cdk5T33 on localization of active Src in A6(1) cells. A6(1) corneal epithelial cells were transiently transfected with EGFP-Cdk5 (A,B) or EGFP- Cdk5T33 (C,D) and then cultured overnight. Cells were immunostained with antibody specific for active Src(pY416) and rhodamine-tagged secondary antibody. (A) EGFP fluorescence showing a cell transfected with EGFP-Cdk5. (B) Rhodamine fluorescence of the same field showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5-transfected cell, active Src is located primarily in the perinuclear region (arrow), with little active Src in cell processes. By contrast, cell processes are well stained in untransfected cells in the same field (arrowhead). (C) EGFP fluorescence of a cell transfected with EGFP-Cdk5T33. (D) Rhodamine fluorescence of the same field, showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5T33- transfected cell, cell processes are well stained, with high concentrations of active Src in cell processes (arrowhead). Active Src is also present in lamellipodia (double arrow). Bar, 25 µm.

Article Snippet: 25-50 μg of total cell extract were immunoblotted as previously described (Gao et al., 1997) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416cSrc (Cell Signaling Technology, Beverly, MA).

Techniques: Transfection, Cell Culture, Staining

Journal: Journal of cell science

Article Title: Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

doi: 10.1242/jcs.01271

Figure Lengend Snippet: Fig. 8. Immunoblotting of Src and active Src in A6(1) cells with and without olomoucine. (A) Multiple scratch wounds were made in confluent cultures of A6(1) cells. Scratch-wounded cultures were incubated in the absence or presence of 15 µM olomoucine and cell lysates were prepared. Next, 50 µg whole cell lysate (WCL) was loaded into each lane of a 12% SDS-polyacrylamide gel and immunoblotted with antibodies against Src or active Src (pY416). (B) The results were quantified by densitometry and averaged (n=5). Olomoucine treatment increased the level of Src(pY416) more than 2.5 times (P=0.04). Error bars=s.e.m. (C) A6(1) cells were transiently transfected with the dominant negative construct EGFP-Cdk5T33. (Left) Whole cell lysate (50 µg) was immunoblotted with antibodies against Src or active Src (pY416). Quantification by densitometry indicated that the level of Src(pY416) was elevated 1.8±0.3 times (n=3) in transiently transfected cells. (Right) 50 µg whole cell extract was immunoblotted with anti- Cdk5 antibody to demonstrate expression of EGFP- Cdk5T33 (single arrow, Cdk5; double arrow, EGFP- Cdk5). (D) A(6)1 cells were incubated in the absence or presence of olomoucine. Cell extracts from subconfluent cultures were immunoprecipitated with anti-Cdk5 antibody and the precipitated proteins were immunoblotted for Src and active Src(pY416) (left) or Cdk5 (right). Both Src and active Src(pY416) were detected in Cdk5 immunoprecipitates (arrow). An unidentified, more rapidly migrating band was occasionally detected by this antibody but does not correspond to Src(pY416) according to manufacturer’s technical data sheet. Immunoblotting with Cdk5 antibody confirmed that immunoprecipitation was effective.

Article Snippet: 25-50 μg of total cell extract were immunoblotted as previously described (Gao et al., 1997) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416cSrc (Cell Signaling Technology, Beverly, MA).

Techniques: Western Blot, Incubation, Transfection, Dominant Negative Mutation, Construct, Expressing, Immunoprecipitation

Journal: Journal of cell science

Article Title: Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

doi: 10.1242/jcs.01271

Figure Lengend Snippet: Fig. 9. Confluent cultures of A6(1) cells were scratch wounded then cultured with or without the Src inhibitor PP1 or the inactive analog PP3. After 24 hours, cultures were double stained with antibody for phosphorylated Cdk5(pY15) (A-C) and rhodamine phalloidin (D-F). (A) In the presence of PP3, Cdk5(pY15) immunofluorescence was observed in a band of cells along the wound edge. (B) In the presence of PP1, little or no Cdk5(pY15) immunofluorescence was detected along the wound edge. (C) Omission of the primary antibody to Cdk5(pY15) showed no detectable immunofluorescence. (D) Rhodamine-phalloidin staining of cells shown in (A) showed polymerization of F-actin along the wound edge, with diffuse cytoplasmic staining and formation of numerous lamellipodia (arrows). (E) Rhodamine-phalloidin staining of cells shown in (B) showed that the actin cytoskeleton was primarily cortical (open arrow) and few lamellipodia were formed. (F) Control cultures incubated without PP3 or PP1 were indistinguishable from those incubated with PP3. Numerous lamellipodia were observed (arrows).

Article Snippet: 25-50 μg of total cell extract were immunoblotted as previously described (Gao et al., 1997) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416cSrc (Cell Signaling Technology, Beverly, MA).

Techniques: Cell Culture, Staining, Control, Incubation

Journal: Molecular Oncology

Article Title: PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met‐addicted tumor cells

doi: 10.1016/j.molonc.2015.07.001

Figure Lengend Snippet: Effect of Met knockdown on PEA3 factors expression. (A) Expression levels of ETV4, ETV5 and ETV1 mRNA were measured by RT‐qPCR in GTL16 and EBC‐1 cells after Met knockdown by specific siRNA. (B) The expression of Met mRNA was detected ...

Article Snippet: The following antibodies were used at a concentration of 1:1000 unless otherwise indicated: Met (Cell Signaling, 3148), phospho‐Met (Cell Signaling, 3126), EGFR (Cell Signaling, 2646), phospho‐EGFR (Cell Signaling, 2236), AKT (Santa Cruz, sc‐8312), phospho‐AKT (Ser473) (Cell Signaling, 9271), ERK (Santa Cruz, sc‐154), phospho‐ERK (Thr202/Tyr204) (Cell Signaling, 9106), ERM/ETV5 antibody (anti‐ERM 12–226 ) ( Baert et al., 1997 ), ER81/ETV1 (Abcam, ab81086, 1/500), PEA3/ETV4 (Santa Cruz, sc‐113), ETS1 (Santa Cruz, sc‐111), ERG (Epitomics, 2085), Actin (Santa Cruz, sc‐47778, 1:250), GAPDH (Santa Cruz, sc‐32233).

Techniques: Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met‐addicted tumor cells

doi: 10.1016/j.molonc.2015.07.001

Figure Lengend Snippet: Consequence of MEK and PI3K inhibition on PEA3 factors expression. (A) Expression levels of ETV4, ETV5, ETV1 and Met mRNA were measured by RT‐qPCR in GTL16 after treatment 24 h with 0.4 μM of Met kinase inhibitor PHA, ...

Article Snippet: The following antibodies were used at a concentration of 1:1000 unless otherwise indicated: Met (Cell Signaling, 3148), phospho‐Met (Cell Signaling, 3126), EGFR (Cell Signaling, 2646), phospho‐EGFR (Cell Signaling, 2236), AKT (Santa Cruz, sc‐8312), phospho‐AKT (Ser473) (Cell Signaling, 9271), ERK (Santa Cruz, sc‐154), phospho‐ERK (Thr202/Tyr204) (Cell Signaling, 9106), ERM/ETV5 antibody (anti‐ERM 12–226 ) ( Baert et al., 1997 ), ER81/ETV1 (Abcam, ab81086, 1/500), PEA3/ETV4 (Santa Cruz, sc‐113), ETS1 (Santa Cruz, sc‐111), ERG (Epitomics, 2085), Actin (Santa Cruz, sc‐47778, 1:250), GAPDH (Santa Cruz, sc‐32233).

Techniques: Inhibition, Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met‐addicted tumor cells

doi: 10.1016/j.molonc.2015.07.001

Figure Lengend Snippet: Consequence of PEA3 knockdown on proliferation, anchorage independent growth and survival. (A, B, C) GTL16 and EBC‐1 cells were transfected with control siRNA (Si‐Ctrl) or siRNA targeted ETV1, ETV4 and ETV5 or Met. (A) Proliferation of ...

Article Snippet: The following antibodies were used at a concentration of 1:1000 unless otherwise indicated: Met (Cell Signaling, 3148), phospho‐Met (Cell Signaling, 3126), EGFR (Cell Signaling, 2646), phospho‐EGFR (Cell Signaling, 2236), AKT (Santa Cruz, sc‐8312), phospho‐AKT (Ser473) (Cell Signaling, 9271), ERK (Santa Cruz, sc‐154), phospho‐ERK (Thr202/Tyr204) (Cell Signaling, 9106), ERM/ETV5 antibody (anti‐ERM 12–226 ) ( Baert et al., 1997 ), ER81/ETV1 (Abcam, ab81086, 1/500), PEA3/ETV4 (Santa Cruz, sc‐113), ETS1 (Santa Cruz, sc‐111), ERG (Epitomics, 2085), Actin (Santa Cruz, sc‐47778, 1:250), GAPDH (Santa Cruz, sc‐32233).

Techniques: Transfection

Journal: Molecular Oncology

Article Title: PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met‐addicted tumor cells

doi: 10.1016/j.molonc.2015.07.001

Figure Lengend Snippet: Consequence of PEA3 knockdown on migration and invasion. (A, B, C) GTL16 and EBC‐1 cells were transfected with control siRNA (Si‐Ctrl) or siRNA targeted ETV1, ETV4 and ETV5 or Met. (A) Migration of GTL16 and EBC‐1 was determined ...

Article Snippet: The following antibodies were used at a concentration of 1:1000 unless otherwise indicated: Met (Cell Signaling, 3148), phospho‐Met (Cell Signaling, 3126), EGFR (Cell Signaling, 2646), phospho‐EGFR (Cell Signaling, 2236), AKT (Santa Cruz, sc‐8312), phospho‐AKT (Ser473) (Cell Signaling, 9271), ERK (Santa Cruz, sc‐154), phospho‐ERK (Thr202/Tyr204) (Cell Signaling, 9106), ERM/ETV5 antibody (anti‐ERM 12–226 ) ( Baert et al., 1997 ), ER81/ETV1 (Abcam, ab81086, 1/500), PEA3/ETV4 (Santa Cruz, sc‐113), ETS1 (Santa Cruz, sc‐111), ERG (Epitomics, 2085), Actin (Santa Cruz, sc‐47778, 1:250), GAPDH (Santa Cruz, sc‐32233).

Techniques: Migration, Transfection