Structured Review

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During EMT, NCAM localizes into lipid rafts together with p59 Fyn and <t>FAK.</t> ( A ) <t>Immunoblotting</t> analysis of NMuMG cells reveals an increase in p59 Fyn phosphorylation upon EMT induction (+TGFβ). Immunoblotting for total p59 Fyn was used as
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1) Product Images from "NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin"

Article Title: NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin

Journal:

doi: 10.1038/emboj.2008.178

During EMT, NCAM localizes into lipid rafts together with p59 Fyn and FAK. ( A ) Immunoblotting analysis of NMuMG cells reveals an increase in p59 Fyn phosphorylation upon EMT induction (+TGFβ). Immunoblotting for total p59 Fyn was used as
Figure Legend Snippet: During EMT, NCAM localizes into lipid rafts together with p59 Fyn and FAK. ( A ) Immunoblotting analysis of NMuMG cells reveals an increase in p59 Fyn phosphorylation upon EMT induction (+TGFβ). Immunoblotting for total p59 Fyn was used as

Techniques Used:

2) Product Images from "FAK-heterozygous mice display enhanced tumour angiogenesis"

Article Title: FAK-heterozygous mice display enhanced tumour angiogenesis

Journal: Nature communications

doi: 10.1038/ncomms3020

Tumour-free angiogenesis is enhanced in FAK-heterozygous mice Sponges implanted into the flanks of either FAK-het mice or WT controls were injected with VEGF or PBS every two days for two weeks. At two weeks post implantation, in vivo angiogenesis was assessed by counting the numbers of blood vessels per mm 2 in sponge sections from WT and FAK-het mice. VEGF-treatment enhanced angiogenesis in both WT and FAK-het mice. However, quantitation showed that angiogenesis is elevated both at the baseline level, PBS-treated, (PBS) and after VEGF-stimulation (VEGF) in FAK-het mice. Upper bar chart represents mean numbers of blood vessels/ mm 2 of sponge section + s.e.m. Lower bar chart shows that the relative fold-increase in microvessel density of FAK-het mice over WT controls was similar for both PBS and VEGF-treated sponges. Arrowheads indicate blood vessels. DAPI-stained nuclei, blue, n=7 WT and n=10 FAK-het mice, Student’s t-test, ns, not statistically significant different, ** P
Figure Legend Snippet: Tumour-free angiogenesis is enhanced in FAK-heterozygous mice Sponges implanted into the flanks of either FAK-het mice or WT controls were injected with VEGF or PBS every two days for two weeks. At two weeks post implantation, in vivo angiogenesis was assessed by counting the numbers of blood vessels per mm 2 in sponge sections from WT and FAK-het mice. VEGF-treatment enhanced angiogenesis in both WT and FAK-het mice. However, quantitation showed that angiogenesis is elevated both at the baseline level, PBS-treated, (PBS) and after VEGF-stimulation (VEGF) in FAK-het mice. Upper bar chart represents mean numbers of blood vessels/ mm 2 of sponge section + s.e.m. Lower bar chart shows that the relative fold-increase in microvessel density of FAK-het mice over WT controls was similar for both PBS and VEGF-treated sponges. Arrowheads indicate blood vessels. DAPI-stained nuclei, blue, n=7 WT and n=10 FAK-het mice, Student’s t-test, ns, not statistically significant different, ** P

Techniques Used: Mouse Assay, Injection, In Vivo, Quantitation Assay, Staining

3) Product Images from "The androgen receptor/filamin A complex as a target in prostate cancer microenvironment"

Article Title: The androgen receptor/filamin A complex as a target in prostate cancer microenvironment

Journal: Cell Death & Disease

doi: 10.1038/s41419-021-03402-7

Androgen triggers AR/β1 integrin/MT-MMP-1 complex assembly and MMP-2 release: a mechanism of CAFs invasion through ECM. AR-positive CAFs pooled from different patients (Table 2 ) were made quiescent. In a , cells were left unchallenged or challenged for 10 min with 10 nM R1881, in the absence or presence of 10 μM enzalutamide (enz) or 10 nM Rh2025u peptide (Rh). Lysate proteins were immune-precipitated using the anti-AR antibody (anti-AR) or control IgG (ctrl IgG). The WB with the indicated antibodies was done to detect proteins in immune-complex. In b , cells were untreated or treated for 24 h as in a . Release of MMP-2 in conditioned media was analyzed by zymography, as described in “Methods” section. In c , lysate proteins were analyzed for FAK activation, using the anti-P-Tyr397FAK antibody. The filter was stripped and re-probed using anti-FAK antibody (upper section). In the lower section, proteins from CAFs lysates were used for Rac pull down assay. The WB with anti-Rac antibody revealed the eluted Rac (Rac-GTP). The total amount of Rac expressed in the corresponding CAFs lysates was also detected. The WB for tubulin expression in CAFs lysate proteins was finally done, as loading control. Results in c are representative of 3 different experiments. In d , androgen stimulation of AR-expressing CAFs triggers the assembly of AR/FlnA/β 1 integrin complex. Such complex is required to activate FAK and Rac, thereby triggering CAFs motility, on one hand. On the other, the AR/FlnA/β 1 integrin complex recruits MT-MMP-1, thus activating pro-MMP2 and the consequent release of MMP-2. Protease-mediated ECM remodeling then follows, thus promoting CAFs invasiveness and their recruitment towards PC cells.
Figure Legend Snippet: Androgen triggers AR/β1 integrin/MT-MMP-1 complex assembly and MMP-2 release: a mechanism of CAFs invasion through ECM. AR-positive CAFs pooled from different patients (Table 2 ) were made quiescent. In a , cells were left unchallenged or challenged for 10 min with 10 nM R1881, in the absence or presence of 10 μM enzalutamide (enz) or 10 nM Rh2025u peptide (Rh). Lysate proteins were immune-precipitated using the anti-AR antibody (anti-AR) or control IgG (ctrl IgG). The WB with the indicated antibodies was done to detect proteins in immune-complex. In b , cells were untreated or treated for 24 h as in a . Release of MMP-2 in conditioned media was analyzed by zymography, as described in “Methods” section. In c , lysate proteins were analyzed for FAK activation, using the anti-P-Tyr397FAK antibody. The filter was stripped and re-probed using anti-FAK antibody (upper section). In the lower section, proteins from CAFs lysates were used for Rac pull down assay. The WB with anti-Rac antibody revealed the eluted Rac (Rac-GTP). The total amount of Rac expressed in the corresponding CAFs lysates was also detected. The WB for tubulin expression in CAFs lysate proteins was finally done, as loading control. Results in c are representative of 3 different experiments. In d , androgen stimulation of AR-expressing CAFs triggers the assembly of AR/FlnA/β 1 integrin complex. Such complex is required to activate FAK and Rac, thereby triggering CAFs motility, on one hand. On the other, the AR/FlnA/β 1 integrin complex recruits MT-MMP-1, thus activating pro-MMP2 and the consequent release of MMP-2. Protease-mediated ECM remodeling then follows, thus promoting CAFs invasiveness and their recruitment towards PC cells.

Techniques Used: Western Blot, Zymography, Activation Assay, Pull Down Assay, Expressing

4) Product Images from "Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma"

Article Title: Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-12-190

Involvement of ERK, FAK, and PI3K/Akt pathways in upregulation of MMP-2 driven by soluble CD147. SMMC-7721 cells were cultured for 30 min (A) and 12 h (B) with 10 μg/ml of E-CD147ECD. CD147 and MMP-2 data were expressed as means ± SEM of three independent determinations of CD147 or MMP-2/α-tubulin ratio. (C) SMMC-7721 cells were treated with E-CD147ECD (10 μg/ml) for 12 h with or without pre-treatment with ERK inhibitor (U0126), FAK inhibitor (FAK inhibitor 14), PI3K inhibitor (LY294002), or combined inhibitors. In A and C, the expression levels of phospho-ERK1/2, ERK1/2, phospho-FAK, FAK, phospho-Akt, Akt, phospho-EGFR, and EGFR were detected by western blotting. Quantitative analysis of phosphorylated fraction relative to the total fraction was shown. Mean values for control groups were normalized to 1. * P
Figure Legend Snippet: Involvement of ERK, FAK, and PI3K/Akt pathways in upregulation of MMP-2 driven by soluble CD147. SMMC-7721 cells were cultured for 30 min (A) and 12 h (B) with 10 μg/ml of E-CD147ECD. CD147 and MMP-2 data were expressed as means ± SEM of three independent determinations of CD147 or MMP-2/α-tubulin ratio. (C) SMMC-7721 cells were treated with E-CD147ECD (10 μg/ml) for 12 h with or without pre-treatment with ERK inhibitor (U0126), FAK inhibitor (FAK inhibitor 14), PI3K inhibitor (LY294002), or combined inhibitors. In A and C, the expression levels of phospho-ERK1/2, ERK1/2, phospho-FAK, FAK, phospho-Akt, Akt, phospho-EGFR, and EGFR were detected by western blotting. Quantitative analysis of phosphorylated fraction relative to the total fraction was shown. Mean values for control groups were normalized to 1. * P

Techniques Used: Cell Culture, Expressing, Western Blot

5) Product Images from "?1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival"

Article Title: ?1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048332

Schematic diagram of laminin-integrin survival signaling in RGC. RGC adhesion to laminin induces integrin activation and initiates integrin signaling. Integrin β1 undergoes a conformational change to an “active” high affinity bound state [83] . Alternatively, in the absence of laminin, or in the context of MMP-9 mediated laminin degradation observed after RIRI in vivo , a β1 integrin stimulatory antibody can bind and maintain integrin β1 in the “active”, signaling conformation at the RGC’s membrane. Activated β1 integrin initiates signal transduction by recruiting the tyrosine protein kinase, FAK. In response to integrin engagement, FAK auto-phosphorylates at Tyr 397 which represents the initial, major step in FAK activation. FAK P-Tyr 397 creates a binding site for Src family kinases. Src recruitment, and activation leads to phosphorylation of multiple amino acids including Tyr 576, 577 required for FAK’s full catalytic activity [37] . PP2 inhibits Src, therefore FAK’s complete activation required for downstream survival signaling. Initial Tyr 397 phosphorylation is also important for recruitment of PI3K. FAK promotes cell survival downstream of laminin and integrin β1 by enhancing PI3K mediated activation of Akt by phosphorylation at Ser 473. Activated Akt is associated with survival [35] , [37] , and inhibition of apoptosis by increasing expression of anti-apoptotic protein bclx L .
Figure Legend Snippet: Schematic diagram of laminin-integrin survival signaling in RGC. RGC adhesion to laminin induces integrin activation and initiates integrin signaling. Integrin β1 undergoes a conformational change to an “active” high affinity bound state [83] . Alternatively, in the absence of laminin, or in the context of MMP-9 mediated laminin degradation observed after RIRI in vivo , a β1 integrin stimulatory antibody can bind and maintain integrin β1 in the “active”, signaling conformation at the RGC’s membrane. Activated β1 integrin initiates signal transduction by recruiting the tyrosine protein kinase, FAK. In response to integrin engagement, FAK auto-phosphorylates at Tyr 397 which represents the initial, major step in FAK activation. FAK P-Tyr 397 creates a binding site for Src family kinases. Src recruitment, and activation leads to phosphorylation of multiple amino acids including Tyr 576, 577 required for FAK’s full catalytic activity [37] . PP2 inhibits Src, therefore FAK’s complete activation required for downstream survival signaling. Initial Tyr 397 phosphorylation is also important for recruitment of PI3K. FAK promotes cell survival downstream of laminin and integrin β1 by enhancing PI3K mediated activation of Akt by phosphorylation at Ser 473. Activated Akt is associated with survival [35] , [37] , and inhibition of apoptosis by increasing expression of anti-apoptotic protein bclx L .

Techniques Used: Activation Assay, In Vivo, Transduction, Binding Assay, Activity Assay, Inhibition, Expressing

The β1 agonist antibody HUTS-21 enhances RGC survival and rescues β1 integrin-FAK signaling in vivo after RIRI. HUTS-21, or isotype control (CTRL) rat antibodies (0.5 mg/ml), or PBS were administered intravitreally (5 µl) in the rat eye twice, 30 minutes, and 2 days after RIRI. A. Immunostaining with a secondary anti-rat fluorescent antibody demonstrating localization of HUTS-21 antibody into the retina 24 hours after RIRI (n = 3 animals were analyzed). Note that HUTS-21 binds to activated β1 receptors on RGC cell body, dendrites, and axons (a, white arrowheads). B. Representative photo-micrographs of retinal flat-mounts of control rats (a), non-treated (RIRI) (b), and intravitreally treated with HUTS-21 (c), IgG control (IgG Ctrl) (d), and vehicle PBS (e) ischemic rats 5 days post-injury (n = 4–6 animals for each group). C. RGC survival was determined by counting FG retrogradely labeled RGCs in flat-mounted retinas 5 days post-RIRI (n = 4–6 animals for each group). Error bars, SD. Student’s t test. **p
Figure Legend Snippet: The β1 agonist antibody HUTS-21 enhances RGC survival and rescues β1 integrin-FAK signaling in vivo after RIRI. HUTS-21, or isotype control (CTRL) rat antibodies (0.5 mg/ml), or PBS were administered intravitreally (5 µl) in the rat eye twice, 30 minutes, and 2 days after RIRI. A. Immunostaining with a secondary anti-rat fluorescent antibody demonstrating localization of HUTS-21 antibody into the retina 24 hours after RIRI (n = 3 animals were analyzed). Note that HUTS-21 binds to activated β1 receptors on RGC cell body, dendrites, and axons (a, white arrowheads). B. Representative photo-micrographs of retinal flat-mounts of control rats (a), non-treated (RIRI) (b), and intravitreally treated with HUTS-21 (c), IgG control (IgG Ctrl) (d), and vehicle PBS (e) ischemic rats 5 days post-injury (n = 4–6 animals for each group). C. RGC survival was determined by counting FG retrogradely labeled RGCs in flat-mounted retinas 5 days post-RIRI (n = 4–6 animals for each group). Error bars, SD. Student’s t test. **p

Techniques Used: In Vivo, Immunostaining, Labeling

Laminin, or β1 integrin activating antibody, HUTS-21, promote integrin signaling in RGCs in vitro. P5 RGCs were cultured on laminin ( a–d ), or PDL ( e–l ) for 48 hours. HUTS-21 ( i, k ) or isotype control ( j, l ) antibodies (1 µg/ml) were added to RGCs cultured on PDL 12 hours after plating. Immunohistochemistry was performed with β1 integrin ( a, e, i, j ), FAK ( b, f ), [pY397]-FAK ( c, g, k, l ), or IgG control antibodies ( d, h ) and analyzed by confocal microscopy. 100–200 cells per condition were analyzed. A representative experiment is shown. A total of N = 5 experiments were performed. Scale bars: 10 µm.
Figure Legend Snippet: Laminin, or β1 integrin activating antibody, HUTS-21, promote integrin signaling in RGCs in vitro. P5 RGCs were cultured on laminin ( a–d ), or PDL ( e–l ) for 48 hours. HUTS-21 ( i, k ) or isotype control ( j, l ) antibodies (1 µg/ml) were added to RGCs cultured on PDL 12 hours after plating. Immunohistochemistry was performed with β1 integrin ( a, e, i, j ), FAK ( b, f ), [pY397]-FAK ( c, g, k, l ), or IgG control antibodies ( d, h ) and analyzed by confocal microscopy. 100–200 cells per condition were analyzed. A representative experiment is shown. A total of N = 5 experiments were performed. Scale bars: 10 µm.

Techniques Used: In Vitro, Cell Culture, Immunohistochemistry, Confocal Microscopy

6) Product Images from "HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion"

Article Title: HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012468

HPK1 associates with SKAP-HOM and negatively regulates Rap1 activation. (A) SKAP-HOM was immunoprecipitated in unstimulated Wehi 231 co/kd cells and pulldowns were detected for SKAP-HOM, HPK1 and RIAM expression by Western blotting; plots are representative for four independent experiments. (B) Left: Active Rap1-GTP of untreated and anti-IgM F(ab')2 (anti µ) stimulated co and kd cells was pulled down with Ral GDS RBD agarose and analysed together with total Rap1 levels by anti-Rap1 Western blotting; quantification of band intensities was performed by normalizing total Rap1 levels of lysates by densitometric evaluation (ImageJ) and all time points of Rap1-GTP samples were compared to the control target at t 0 = 1; GDP/GTP: negative/positive control; right: graph representative for three independently performed experiments. (C) F-actin levels (means ± SD; Student's t; n = 6) of untreated co and kd cells measured by Phalloidin-FITC flow cytometric staining. (D) F-actin levels (means ± SD; Student's t; n = 6) of anti-IgM F(ab')2 (anti µ) stimulated co and kd cells. (E) Untreated and anti-IgM stimulated FAK immunoprecipitates of Wehi 231 co and kd cells were detected for total FAK and pFAK379 expression by Western blotting (n = 3); ns, not significant; pd, pulldown.
Figure Legend Snippet: HPK1 associates with SKAP-HOM and negatively regulates Rap1 activation. (A) SKAP-HOM was immunoprecipitated in unstimulated Wehi 231 co/kd cells and pulldowns were detected for SKAP-HOM, HPK1 and RIAM expression by Western blotting; plots are representative for four independent experiments. (B) Left: Active Rap1-GTP of untreated and anti-IgM F(ab')2 (anti µ) stimulated co and kd cells was pulled down with Ral GDS RBD agarose and analysed together with total Rap1 levels by anti-Rap1 Western blotting; quantification of band intensities was performed by normalizing total Rap1 levels of lysates by densitometric evaluation (ImageJ) and all time points of Rap1-GTP samples were compared to the control target at t 0 = 1; GDP/GTP: negative/positive control; right: graph representative for three independently performed experiments. (C) F-actin levels (means ± SD; Student's t; n = 6) of untreated co and kd cells measured by Phalloidin-FITC flow cytometric staining. (D) F-actin levels (means ± SD; Student's t; n = 6) of anti-IgM F(ab')2 (anti µ) stimulated co and kd cells. (E) Untreated and anti-IgM stimulated FAK immunoprecipitates of Wehi 231 co and kd cells were detected for total FAK and pFAK379 expression by Western blotting (n = 3); ns, not significant; pd, pulldown.

Techniques Used: Activation Assay, Immunoprecipitation, Expressing, Western Blot, Positive Control, Flow Cytometry, Staining

7) Product Images from "PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility"

Article Title: PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

Journal: Biology Open

doi: 10.1242/bio.015883

PTP1B promotes early integrin-dependent FAK activation. WT (A-I) and KO cells (J-R) plated on fibronectin for 5, 10 and 30 min. Cells were double-immunolabeled for FAK and FAK-pY397. (A-F) WT cells showing FAK-pY397 accumulation in a peripheral ring of puncta at 5 and 10 min, and in elongated peripheral adhesions at 30 min (yellow arrowheads). (G-I) Enlarged views (4×) of the boxed regions in C and D. (J-O) KO cells did not show FAK-pY397 accumulation at the cell periphery at 5 min post-plating, but localized in discrete peripheral puncta by 10 min (M) and in elongated peripheral adhesions by 30 min (O). (P-R) Enlarged views of the boxed regions in L and M. (S,T) Quantification of peripheral FAK-pY397 (S, n =42 cells) and FAK (T, n =32 cells) signal as described in Fig. 1 . Differences between mean values at the peak were statistically significant P
Figure Legend Snippet: PTP1B promotes early integrin-dependent FAK activation. WT (A-I) and KO cells (J-R) plated on fibronectin for 5, 10 and 30 min. Cells were double-immunolabeled for FAK and FAK-pY397. (A-F) WT cells showing FAK-pY397 accumulation in a peripheral ring of puncta at 5 and 10 min, and in elongated peripheral adhesions at 30 min (yellow arrowheads). (G-I) Enlarged views (4×) of the boxed regions in C and D. (J-O) KO cells did not show FAK-pY397 accumulation at the cell periphery at 5 min post-plating, but localized in discrete peripheral puncta by 10 min (M) and in elongated peripheral adhesions by 30 min (O). (P-R) Enlarged views of the boxed regions in L and M. (S,T) Quantification of peripheral FAK-pY397 (S, n =42 cells) and FAK (T, n =32 cells) signal as described in Fig. 1 . Differences between mean values at the peak were statistically significant P

Techniques Used: Activation Assay, Immunolabeling

Myosin inhibition restores lamellipodium and integrin-dependent signaling at the periphery of KO cells. KO cells were incubated with blebbistatin (+bleb) or vehicle (control) and plated for 10 min on fibronectin. Control cells (A) or +bleb cells (B) were labeled with Phalloidin-TRITC. (C) Quantification of fluorescence in line scans ( n =30 cells). Control cells (D) or +bleb cells (E) labeled with anti-FAK-pY397. (F) Quantification of FAK-pY397 fluorescence signal ( n =40 cells). Control cells (G) or +bleb cells (H) labeled with anti-Src-pY418. (I) Quantification of Src-pY418 signal ( n =74 cells). Control cells (J) or +bleb cells (K) labeled with anti-paxillin-pY118. (L) Quantification of paxillin-pY118 signal ( n =40 cells). The robust effect of blebbistatin on the distribution of F-actin, FAK-pY397, Src-pY418 and paxillin-pY118 is better appreciated in enlarged views (3×) of peripheral regions (yellow boxes). In all cases differences between mean values at the peak were statistically significant P
Figure Legend Snippet: Myosin inhibition restores lamellipodium and integrin-dependent signaling at the periphery of KO cells. KO cells were incubated with blebbistatin (+bleb) or vehicle (control) and plated for 10 min on fibronectin. Control cells (A) or +bleb cells (B) were labeled with Phalloidin-TRITC. (C) Quantification of fluorescence in line scans ( n =30 cells). Control cells (D) or +bleb cells (E) labeled with anti-FAK-pY397. (F) Quantification of FAK-pY397 fluorescence signal ( n =40 cells). Control cells (G) or +bleb cells (H) labeled with anti-Src-pY418. (I) Quantification of Src-pY418 signal ( n =74 cells). Control cells (J) or +bleb cells (K) labeled with anti-paxillin-pY118. (L) Quantification of paxillin-pY118 signal ( n =40 cells). The robust effect of blebbistatin on the distribution of F-actin, FAK-pY397, Src-pY418 and paxillin-pY118 is better appreciated in enlarged views (3×) of peripheral regions (yellow boxes). In all cases differences between mean values at the peak were statistically significant P

Techniques Used: Inhibition, Incubation, Labeling, Fluorescence

PTP1B regulates cell contractility and spreading. (A) In WT cells, PTP1B cooperate with β3 integrin to activate Src/FAK signaling and repress RhoA-myosin activation (dotted lines and boxes). These events modulate negatively acto-myosin contractility at the cell cortex, facilitating the assembly of a lamellipodium (LP) and peripheral adhesions required for cell spreading. Impairing Src/FAK signaling pathway by dominant negative Src (SrcDN) and FRNK expression, or increasing RhoA function by expression of RhoA L63, induces a KO cell phenotype. In WT cells, phosphorylation of paxillin promotes Rac1 activity. (B) In KO cells, early integrin-dependent Src/FAK activation is impaired (dotted box). RhoA/myosin-dependent contractility is enhanced (↑), and as a consequence, filamin and FilGAP availability likely undermine integrin function and lamellipodium assembly, respectively. This condition facilitates cell contraction. Expression of constitutive active Src (SrcYF), or incubation with blebbistatin, restores peripheral adhesions and the lamellipodium. In KO cells Rac1 activity is not induced efficiently, likely by reduced phosphorylation of paxillin and other adaptors, and by negative regulation imposed by enhanced RhoA/myosin signaling. Expression of constitutively active Rac1 L61 rescues lamellipodium formation. Expression of the phosphomimetic paxillin Y31E/Y118E is not enough for adhesion assembly.
Figure Legend Snippet: PTP1B regulates cell contractility and spreading. (A) In WT cells, PTP1B cooperate with β3 integrin to activate Src/FAK signaling and repress RhoA-myosin activation (dotted lines and boxes). These events modulate negatively acto-myosin contractility at the cell cortex, facilitating the assembly of a lamellipodium (LP) and peripheral adhesions required for cell spreading. Impairing Src/FAK signaling pathway by dominant negative Src (SrcDN) and FRNK expression, or increasing RhoA function by expression of RhoA L63, induces a KO cell phenotype. In WT cells, phosphorylation of paxillin promotes Rac1 activity. (B) In KO cells, early integrin-dependent Src/FAK activation is impaired (dotted box). RhoA/myosin-dependent contractility is enhanced (↑), and as a consequence, filamin and FilGAP availability likely undermine integrin function and lamellipodium assembly, respectively. This condition facilitates cell contraction. Expression of constitutive active Src (SrcYF), or incubation with blebbistatin, restores peripheral adhesions and the lamellipodium. In KO cells Rac1 activity is not induced efficiently, likely by reduced phosphorylation of paxillin and other adaptors, and by negative regulation imposed by enhanced RhoA/myosin signaling. Expression of constitutively active Rac1 L61 rescues lamellipodium formation. Expression of the phosphomimetic paxillin Y31E/Y118E is not enough for adhesion assembly.

Techniques Used: Activation Assay, Dominant Negative Mutation, Expressing, Activity Assay, Incubation

Src-FAK-signaling and RhoA downregulation promote adhesion formation. In all conditions cells were plated on fibronectin for 10 min. (A-C) KO cells expressing constitutively active SrcY529F-HA were immunolabeled for paxillin-pY118 and HA. Transfected cells restored paxillin-pY118 accumulation at peripheral puncta (white arrow). (D) Quantification of paxillin-pY118 signal as described in Fig. 1 . Transfected (T=29 cells, green line) and non transfected (NT=64 cells, red line) cells. (E-L) WT cells expressing SrcKD/Y529F-HA mutant, to inhibit Src-dependent signaling (E-H), or myc-FRNK, to inhibit FAK function (I-L). Cells were immunolabeled for paxillin-pY118 (red signal) and HA/myc tags (green signals). Transfected WT cells (white arrows) show a significant reduction of paxillin-pY118 accumulation at cell margins compared to non transfected cells. (H,L) Quantification of paxillin-pY118 signal (T=30 cells, NT=23 cells). (M-T) WT cells expressing SrcKD/Y529F-HA (M-P) or myc-FRNK (Q-T) were incubated with blebbistatin and plated in the presence of the drug. Cells were immunolabeled for paxillin-pY118. Note that transfected cells (white arrows) display similar paxillin-pY118 accumulation at the periphery as non transfected cells. (P,T) Quantification of paxillin-pY118 signals (T=25 cells, NT=28 cells). (U-X) WT cells expressing active myc-RhoA L63 were immunolabeled for myc and Src-pY418. Transfected cells (white arrows) show significant reduction of Src-pY418 signal at the cell periphery. (X) Quantification of Src-pY418 signal (T=24 cells, NT=22 cells). (D,H,L,X) Differences between mean values at the peak were statistically significant P
Figure Legend Snippet: Src-FAK-signaling and RhoA downregulation promote adhesion formation. In all conditions cells were plated on fibronectin for 10 min. (A-C) KO cells expressing constitutively active SrcY529F-HA were immunolabeled for paxillin-pY118 and HA. Transfected cells restored paxillin-pY118 accumulation at peripheral puncta (white arrow). (D) Quantification of paxillin-pY118 signal as described in Fig. 1 . Transfected (T=29 cells, green line) and non transfected (NT=64 cells, red line) cells. (E-L) WT cells expressing SrcKD/Y529F-HA mutant, to inhibit Src-dependent signaling (E-H), or myc-FRNK, to inhibit FAK function (I-L). Cells were immunolabeled for paxillin-pY118 (red signal) and HA/myc tags (green signals). Transfected WT cells (white arrows) show a significant reduction of paxillin-pY118 accumulation at cell margins compared to non transfected cells. (H,L) Quantification of paxillin-pY118 signal (T=30 cells, NT=23 cells). (M-T) WT cells expressing SrcKD/Y529F-HA (M-P) or myc-FRNK (Q-T) were incubated with blebbistatin and plated in the presence of the drug. Cells were immunolabeled for paxillin-pY118. Note that transfected cells (white arrows) display similar paxillin-pY118 accumulation at the periphery as non transfected cells. (P,T) Quantification of paxillin-pY118 signals (T=25 cells, NT=28 cells). (U-X) WT cells expressing active myc-RhoA L63 were immunolabeled for myc and Src-pY418. Transfected cells (white arrows) show significant reduction of Src-pY418 signal at the cell periphery. (X) Quantification of Src-pY418 signal (T=24 cells, NT=22 cells). (D,H,L,X) Differences between mean values at the peak were statistically significant P

Techniques Used: Expressing, Immunolabeling, Transfection, Mutagenesis, Incubation

8) Product Images from "Functional Activation of Src Family Kinase Yes Protein Is Essential for the Enhanced Malignant Properties of Human Melanoma Cells Expressing Ganglioside GD3 *"

Article Title: Functional Activation of Src Family Kinase Yes Protein Is Essential for the Enhanced Malignant Properties of Human Melanoma Cells Expressing Ganglioside GD3 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.164798

Effects of the knockdown of p130Cas, paxillin, or FAK on phosphorylation levels of Yes. A , the effects of the knockdown of p130Cas or paxillin on the phosphorylation levels of Yes in G5 were examined by immunoblotting ( IB ). The tyrosine phosphorylation
Figure Legend Snippet: Effects of the knockdown of p130Cas, paxillin, or FAK on phosphorylation levels of Yes. A , the effects of the knockdown of p130Cas or paxillin on the phosphorylation levels of Yes in G5 were examined by immunoblotting ( IB ). The tyrosine phosphorylation

Techniques Used:

9) Product Images from "Quantitative in vivo imaging of the effects of inhibiting integrin signalling via Src and FAK on cancer cell movement; effects on E-cadherin dynamics"

Article Title: Quantitative in vivo imaging of the effects of inhibiting integrin signalling via Src and FAK on cancer cell movement; effects on E-cadherin dynamics

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-1454

Inhibition of Src-dependent phosphorylation of FAK downstream of β1-integrin disrupts E-cadherin endocytosis and strengthens cell-cell junctions. ( A ) Number of single cells that disaggregate from a dispase treated monolayer. Values represent the
Figure Legend Snippet: Inhibition of Src-dependent phosphorylation of FAK downstream of β1-integrin disrupts E-cadherin endocytosis and strengthens cell-cell junctions. ( A ) Number of single cells that disaggregate from a dispase treated monolayer. Values represent the

Techniques Used: Inhibition

10) Product Images from "Cross Talk between the TM4SF5/Focal Adhesion Kinase and the Interleukin-6/STAT3 Pathways Promotes Immune Escape of Human Liver Cancer Cells"

Article Title: Cross Talk between the TM4SF5/Focal Adhesion Kinase and the Interleukin-6/STAT3 Pathways Promotes Immune Escape of Human Liver Cancer Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00660-14

Inhibition of FAK activity did not alter IL-6-mediated pY 705 STAT3 in Chang or SNU761 cells. The cells were manipulated as explained in the legend to and were treated with or without PF271 (a specific FAK inhibitor) prior to being kept in suspension
Figure Legend Snippet: Inhibition of FAK activity did not alter IL-6-mediated pY 705 STAT3 in Chang or SNU761 cells. The cells were manipulated as explained in the legend to and were treated with or without PF271 (a specific FAK inhibitor) prior to being kept in suspension

Techniques Used: Inhibition, Activity Assay

Suppression of STAT3/JAK signaling inhibited IL-6-mediated FAK phosphorylation of Chang-TM4SF5 cells but did not recover IL-6-suppressed FAK phosphorylation of SNU761-TM4SF5 cells. Cells were manipulated, as explained in the legend to , after transfection
Figure Legend Snippet: Suppression of STAT3/JAK signaling inhibited IL-6-mediated FAK phosphorylation of Chang-TM4SF5 cells but did not recover IL-6-suppressed FAK phosphorylation of SNU761-TM4SF5 cells. Cells were manipulated, as explained in the legend to , after transfection

Techniques Used: Transfection

Cross talk between the TM4SF5/FAK and IL-6/STAT3 pathways. In normal and cancerous hepatocytes lacking TM4SF5 expression, IL-6 treatment causes FAK activation (not depicted). TM4SF5-expressing cancer cells exhibited IL-6-independent and -dependent STAT3
Figure Legend Snippet: Cross talk between the TM4SF5/FAK and IL-6/STAT3 pathways. In normal and cancerous hepatocytes lacking TM4SF5 expression, IL-6 treatment causes FAK activation (not depicted). TM4SF5-expressing cancer cells exhibited IL-6-independent and -dependent STAT3

Techniques Used: Expressing, Activation Assay

11) Product Images from "FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion"

Article Title: FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200809110

FAK regulates a switch in tyrosine phosphorylation at focal adhesions and invadopodia. (A) Cell lysates from MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were analyzed by immunoblotting (IB) or immunoprecipitation (IP)/immunoblotting and probed with phospho-specific antibodies anti-pY397 FAK, anti-pY416 Src, anti-pY410 p130Cas, anti-pY31 paxillin, anti-pY421 cortactin, or antiphosphotyrosine and antibodies to total proteins anti-FAK, anti-Src, anti-p130Cas, antipaxillin, anticortactin, or anti-Tks5/FISH. (B) Quantification of immunoblots or immunoprecipitation/immunoblots is expressed as fold change in phosphorylation to total protein. Fold change was determined by the ratio of normalized phosphorylation to total protein in FAK siRNA compared with control siRNA. Data shown are means ± SEM of three independent experiments.
Figure Legend Snippet: FAK regulates a switch in tyrosine phosphorylation at focal adhesions and invadopodia. (A) Cell lysates from MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were analyzed by immunoblotting (IB) or immunoprecipitation (IP)/immunoblotting and probed with phospho-specific antibodies anti-pY397 FAK, anti-pY416 Src, anti-pY410 p130Cas, anti-pY31 paxillin, anti-pY421 cortactin, or antiphosphotyrosine and antibodies to total proteins anti-FAK, anti-Src, anti-p130Cas, antipaxillin, anticortactin, or anti-Tks5/FISH. (B) Quantification of immunoblots or immunoprecipitation/immunoblots is expressed as fold change in phosphorylation to total protein. Fold change was determined by the ratio of normalized phosphorylation to total protein in FAK siRNA compared with control siRNA. Data shown are means ± SEM of three independent experiments.

Techniques Used: Transfection, Immunoprecipitation, Fluorescence In Situ Hybridization, Western Blot

FAK negatively regulates invadopodia formation and dynamics. (A) Cell lysates from MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi A and FAKsi B) were analyzed by Western blotting and probed for FAK or Pyk2. Actin was probed as a loading control. (B) MTLn3 cells were plated on fibronectin-coated coverslips and stained with anticortactin antibody (green) and rhodamine-phalloidin (red). Boxed regions depict regions of invadopodia shown magnified in insets. (C) Quantification of cortactin- and actin-containing invadopodia is expressed as the mean number of invadopodia per cell. (D) GFP-cortactin was transiently cotransfected with control siRNA or FAK siRNA (FAK siRNA B is shown) into MTLn3 cells. Cells were plated on fibronectin-coated glass-bottomed dishes and analyzed by time-lapse fluorescence microscopy. Time-lapse montages demonstrate representative images of the dynamics of the invadopodia marker GFP-cortactin over a period of 10 min. (E) Rate constants for assembly and disassembly were calculated from plots of fluorescence intensities of GFP-cortactin as described in Materials and methods ( Videos 1–3 ). (F) Representative x-z confocal images of invadopodia in MTLn3 cells show cortactin-containing invadopodia (red) projecting into the gelatin matrix (green). Arrows indicate representative invadopodia. Data shown are means ± SEM of three independent experiments. *, P
Figure Legend Snippet: FAK negatively regulates invadopodia formation and dynamics. (A) Cell lysates from MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi A and FAKsi B) were analyzed by Western blotting and probed for FAK or Pyk2. Actin was probed as a loading control. (B) MTLn3 cells were plated on fibronectin-coated coverslips and stained with anticortactin antibody (green) and rhodamine-phalloidin (red). Boxed regions depict regions of invadopodia shown magnified in insets. (C) Quantification of cortactin- and actin-containing invadopodia is expressed as the mean number of invadopodia per cell. (D) GFP-cortactin was transiently cotransfected with control siRNA or FAK siRNA (FAK siRNA B is shown) into MTLn3 cells. Cells were plated on fibronectin-coated glass-bottomed dishes and analyzed by time-lapse fluorescence microscopy. Time-lapse montages demonstrate representative images of the dynamics of the invadopodia marker GFP-cortactin over a period of 10 min. (E) Rate constants for assembly and disassembly were calculated from plots of fluorescence intensities of GFP-cortactin as described in Materials and methods ( Videos 1–3 ). (F) Representative x-z confocal images of invadopodia in MTLn3 cells show cortactin-containing invadopodia (red) projecting into the gelatin matrix (green). Arrows indicate representative invadopodia. Data shown are means ± SEM of three independent experiments. *, P

Techniques Used: Transfection, Western Blot, Staining, Fluorescence, Microscopy, Marker

FAK modulates the localization of pY31-paxillin, pY410 p130Cas, and Tks5 in MTLn3 cells. (A–F) MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were cultured on fibronectin-gelatin coverslips and stained with anti-pY31 paxillin (A and B), anti-pY410 p130Cas (C and D), or anti-Tks5/FISH antibody (E and F; red). Cells were costained with the invadopodia marker cortactin (A, C, and E; green) or the focal adhesion marker vinculin (B, D, and F; green). Regions outlined by boxes correspond to magnified images of pY31-paxillin, pY410 p130Cas, or Tks5 localization at focal adhesions and/or invadopodia shown in insets. Bars, 10 µm.
Figure Legend Snippet: FAK modulates the localization of pY31-paxillin, pY410 p130Cas, and Tks5 in MTLn3 cells. (A–F) MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were cultured on fibronectin-gelatin coverslips and stained with anti-pY31 paxillin (A and B), anti-pY410 p130Cas (C and D), or anti-Tks5/FISH antibody (E and F; red). Cells were costained with the invadopodia marker cortactin (A, C, and E; green) or the focal adhesion marker vinculin (B, D, and F; green). Regions outlined by boxes correspond to magnified images of pY31-paxillin, pY410 p130Cas, or Tks5 localization at focal adhesions and/or invadopodia shown in insets. Bars, 10 µm.

Techniques Used: Transfection, Cell Culture, Staining, Fluorescence In Situ Hybridization, Marker

12) Product Images from "Full activity of the deleted in liver cancer 1 (DLC1) tumor suppressor depends on an LD-like motif that binds talin and focal adhesion kinase (FAK)"

Article Title: Full activity of the deleted in liver cancer 1 (DLC1) tumor suppressor depends on an LD-like motif that binds talin and focal adhesion kinase (FAK)

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1112122108

The DLC1 targets talin and FAK contribute to cell migration and anchorage-independent growth. ( A ) Verification of siRNA knockdown in NSCLC line H1299. The specific siRNA knockdown of DLC1, talin, and FAK was confirmed by immunoprecipitation/IB of anti-DLC1, anti-talin, and anti-FAK blots. The Rho-GTP level was also analyzed, and the totalt Rho-immunoblo is shown as loading control. ( B ) Effect of indicated siRNA on cell migration. H1299 cells were analyzed by wound healing and transwell assays 48 h after siRNA transfection. The quantitation of migrated cells in the transwell assay is shown. ( C ) Effect of indicated siRNA on anchorage-independent cell growth. Equal numbers of siRNA-transfected H1299 cells were seeded in soft agar for growth and quantitation.
Figure Legend Snippet: The DLC1 targets talin and FAK contribute to cell migration and anchorage-independent growth. ( A ) Verification of siRNA knockdown in NSCLC line H1299. The specific siRNA knockdown of DLC1, talin, and FAK was confirmed by immunoprecipitation/IB of anti-DLC1, anti-talin, and anti-FAK blots. The Rho-GTP level was also analyzed, and the totalt Rho-immunoblo is shown as loading control. ( B ) Effect of indicated siRNA on cell migration. H1299 cells were analyzed by wound healing and transwell assays 48 h after siRNA transfection. The quantitation of migrated cells in the transwell assay is shown. ( C ) Effect of indicated siRNA on anchorage-independent cell growth. Equal numbers of siRNA-transfected H1299 cells were seeded in soft agar for growth and quantitation.

Techniques Used: Migration, Immunoprecipitation, Transfection, Quantitation Assay, Transwell Assay

DLC1 binds FAK and interferes with paxillin binding to the FAT domain of FAK: dependence on the LD-like motif. ( A ) Endogenous DLC1 and FAK form a complex in human cell lines. A skin fibroblast line (Skin) and NSCLC line H1703 were analyzed by reciprocal coimmunoprecipitation. ( B ) Colocalization of DLC1 with FAK in NSCLC lines. Endogenous DLC1 in H1703 cells or transfected DLC1 in H358 cells were stained with anti-DLC1 antibody (red) and endogenous FAK with anti-FAK antibodies (green). The confocal images are representative of the majority of cells observed. (Scale bar: 10 μm.) ( C ) Association of DLC1 with talin or FAK depends on the LD-like motif. Pull down of endogenous talin and FAK by GST-fusion DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. ( D ) Interaction between DLC1 and FAK is talin-independent. Pull-down of 293T cells, with or without talin siRNA treatment, of endogenous FAK and talin by DLC1 GST (448–500) WT or delLD. ( E ) DLC1-FAK interaction is through the FAT-domain of FAK and requires the LD-like motif of DLC1. Pull down of GFP-FAT, derived from FAK, by GST-tagged DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. ( F ) DLC1 can compete with paxillin but not with talin for binding the FAT-domain. GST-FAT was cotransfected with increasing amounts of DLC1 at the indicated ratios in 293T cells. The pull down of DLC1, endogenous paxillin, and endogenous talin are shown, and the transfected proteins are also shown. ( G ) Schematic representation of the similar region in the FAT domain of FAK binds paxillin and the LD-like motif, whereas talin binds a distinct region of the FAT domain as well as the LD-like domain.
Figure Legend Snippet: DLC1 binds FAK and interferes with paxillin binding to the FAT domain of FAK: dependence on the LD-like motif. ( A ) Endogenous DLC1 and FAK form a complex in human cell lines. A skin fibroblast line (Skin) and NSCLC line H1703 were analyzed by reciprocal coimmunoprecipitation. ( B ) Colocalization of DLC1 with FAK in NSCLC lines. Endogenous DLC1 in H1703 cells or transfected DLC1 in H358 cells were stained with anti-DLC1 antibody (red) and endogenous FAK with anti-FAK antibodies (green). The confocal images are representative of the majority of cells observed. (Scale bar: 10 μm.) ( C ) Association of DLC1 with talin or FAK depends on the LD-like motif. Pull down of endogenous talin and FAK by GST-fusion DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. ( D ) Interaction between DLC1 and FAK is talin-independent. Pull-down of 293T cells, with or without talin siRNA treatment, of endogenous FAK and talin by DLC1 GST (448–500) WT or delLD. ( E ) DLC1-FAK interaction is through the FAT-domain of FAK and requires the LD-like motif of DLC1. Pull down of GFP-FAT, derived from FAK, by GST-tagged DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. ( F ) DLC1 can compete with paxillin but not with talin for binding the FAT-domain. GST-FAT was cotransfected with increasing amounts of DLC1 at the indicated ratios in 293T cells. The pull down of DLC1, endogenous paxillin, and endogenous talin are shown, and the transfected proteins are also shown. ( G ) Schematic representation of the similar region in the FAT domain of FAK binds paxillin and the LD-like motif, whereas talin binds a distinct region of the FAT domain as well as the LD-like domain.

Techniques Used: Binding Assay, Transfection, Staining, Derivative Assay

13) Product Images from "Dominant Suppression of β1 Integrin by Ectopic CD98-ICD Inhibits Hepatocellular Carcinoma Progression"

Article Title: Dominant Suppression of β1 Integrin by Ectopic CD98-ICD Inhibits Hepatocellular Carcinoma Progression

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17111882

CD98-ICD inhibits β1-integrin signaling. ( A ) Co-localization of β1-integrin and CD98-ICD in SMMC-7721 and Huh-7 cells. The co-localization of β1-integrin (red) and CD98 (green) was first analyzed as a positive control ( upper panel). Then, after cells were transfected with eGFP-N1 ( middle panel) or CD98-ICD-EGFP ( lower panel) for 36 h, β1-integrin was visualized to analyze co-localization with CD98-ICD. (Pearson’s coefficient is indicated as numerical data on the right of each panel, n > 3). Nuclei: blue. Bar, 10 µm; ( B ) Co-IP analyses of β1-integrin and CD98-ICD interaction in SMMC-7721 cells; ( C ) changes in the molecular expression patterns were detected in SMMC-7721 and Huh-7 cells transfected with EGFP-N1 or CD98-ICD-EGFP. Western blot scanning densitometry for three independent experiments is shown on the right. Blots were probed for β1-integrin, AKT, or FAK independently to ensure equal protein loading. * p
Figure Legend Snippet: CD98-ICD inhibits β1-integrin signaling. ( A ) Co-localization of β1-integrin and CD98-ICD in SMMC-7721 and Huh-7 cells. The co-localization of β1-integrin (red) and CD98 (green) was first analyzed as a positive control ( upper panel). Then, after cells were transfected with eGFP-N1 ( middle panel) or CD98-ICD-EGFP ( lower panel) for 36 h, β1-integrin was visualized to analyze co-localization with CD98-ICD. (Pearson’s coefficient is indicated as numerical data on the right of each panel, n > 3). Nuclei: blue. Bar, 10 µm; ( B ) Co-IP analyses of β1-integrin and CD98-ICD interaction in SMMC-7721 cells; ( C ) changes in the molecular expression patterns were detected in SMMC-7721 and Huh-7 cells transfected with EGFP-N1 or CD98-ICD-EGFP. Western blot scanning densitometry for three independent experiments is shown on the right. Blots were probed for β1-integrin, AKT, or FAK independently to ensure equal protein loading. * p

Techniques Used: Positive Control, Transfection, Co-Immunoprecipitation Assay, Expressing, Western Blot

14) Product Images from "Thioredoxin Reductase Linked to Cytoskeleton by Focal Adhesion Kinase Reverses Actin S-Nitrosylation and Restores Neutrophil ?2 Integrin Function *"

Article Title: Thioredoxin Reductase Linked to Cytoskeleton by Focal Adhesion Kinase Reverses Actin S-Nitrosylation and Restores Neutrophil ?2 Integrin Function *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.355875

TrxR immunoprecipitation Coomassie-stained gel. The figure shows the band pattern for lysates of air-exposed, control, and HBO 2 -exposed neutrophil lysates immunoprecipitated using anti-TrxR. Proteins listed were identified by MS/MS spectra. For nitric-oxide synthase-2 ( iNOS ) 36 peptides were identified spanning 32% of the protein; for FAK, 12 peptides were identified spanning 9% of the protein; for heat shock protein-90α ( HSP90 α), 20 peptides were identified spanning 24% of the protein; for heat shock protein-90β ( HSP90 β), 22 peptides were identified spanning 34% of the protein; for heat shock cognate 71-kDa protein, 19 peptides were identified spanning 29% of the protein; for TrxR, 10 peptides were identified spanning 18% of the protein; for protein-disulfide isomerase A6 precursor, seven peptides were identified spanning 18% of the protein; for actin, 26 peptides were identified spanning 49% of the protein; for tropomyosin α-1 isoform, seven peptides were identified spanning 26% of the protein; for 14-3-3 protein ζ/δ, 12 peptides were identified spanning 44% of the protein; for Ras-related protein Rab-27B, seven peptides were identified spanning 42% of the protein.
Figure Legend Snippet: TrxR immunoprecipitation Coomassie-stained gel. The figure shows the band pattern for lysates of air-exposed, control, and HBO 2 -exposed neutrophil lysates immunoprecipitated using anti-TrxR. Proteins listed were identified by MS/MS spectra. For nitric-oxide synthase-2 ( iNOS ) 36 peptides were identified spanning 32% of the protein; for FAK, 12 peptides were identified spanning 9% of the protein; for heat shock protein-90α ( HSP90 α), 20 peptides were identified spanning 24% of the protein; for heat shock protein-90β ( HSP90 β), 22 peptides were identified spanning 34% of the protein; for heat shock cognate 71-kDa protein, 19 peptides were identified spanning 29% of the protein; for TrxR, 10 peptides were identified spanning 18% of the protein; for protein-disulfide isomerase A6 precursor, seven peptides were identified spanning 18% of the protein; for actin, 26 peptides were identified spanning 49% of the protein; for tropomyosin α-1 isoform, seven peptides were identified spanning 26% of the protein; for 14-3-3 protein ζ/δ, 12 peptides were identified spanning 44% of the protein; for Ras-related protein Rab-27B, seven peptides were identified spanning 42% of the protein.

Techniques Used: Immunoprecipitation, Staining, Mass Spectrometry

15) Product Images from "?1 Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy"

Article Title: ?1 Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI61350

Perturbed FAK/JNK1 signaling by β 1 integrin inhibition causes tumor cell radiosensitization.
Figure Legend Snippet: Perturbed FAK/JNK1 signaling by β 1 integrin inhibition causes tumor cell radiosensitization.

Techniques Used: Inhibition

Disassembly of FAK/cortactin interaction upon β 1 integrin inhibition.
Figure Legend Snippet: Disassembly of FAK/cortactin interaction upon β 1 integrin inhibition.

Techniques Used: Inhibition

16) Product Images from "MiR-7a is an important mediator in Fas-associated protein with death domain (FADD)-regulated expression of focal adhesion kinase (FAK)"

Article Title: MiR-7a is an important mediator in Fas-associated protein with death domain (FADD)-regulated expression of focal adhesion kinase (FAK)

Journal: Oncotarget

doi: 10.18632/oncotarget.9838

MiR-7a was a mediator in FADD-regulated expression of FAK A. Firstly, transfected FADD siRNA (siFADD) alone or co-transfected with FADD siRNA (siFADD) and miR-7a mimic or inhibitor into B16F10 cells for 48 hours. Then a 24 h period scratch wound healing assay had been launched in those transfected B16F10 cells after the transfection. Inhibiting miR-7a expression rescued FADD interference reduced cell migration. Migration was retarded even more when miR-7a mimic were used instead of inhibitor co-transfected with FADD siRNAs. B. Firstly transfected pRK5-FADD plasmid alone or co-transfected pRK5-FADD plasmid together with miR-7a mimic or inhibitor into B16F1 cells for 48 hours. Then a 24 h period scratch wound healing assay had been launched in those B16F1 cells after the transfection. FADD overexpression promoted cell migration which is repressible by miR-7a mimic. Cells co-transfected with pRK5-FADD and miR-7a inhibitor exhibited the fastest migration rate among the four different samples. C. and D. B16F1 melanoma cells treated with pRK5 empty vector plasmids, pRK5-FADD plasmids, pRK5-FADD plasmids plus miR-7a mimic and pRK5-FADD plasmids plus miR-7a inhibitor, were collected and suspended in PBS. Then the treated cells were injected into the tail vein of C57BL/6 mice. After 14 days, mice were sacrificed with all the lung tissues were excised and the surface lung tumor nodules were counted. The representative pictures of lung metastases (C) and the number of metastasis foci on the surface of lung (D). Data are presented as mean ± S.D. n=5. E, F. and G. Representative western blot analysis (E) and quantification of expression of FADD (F) and FAK (G) in the miR-7a inhibitor/mimic co-transfected with FADD siRNAs experiment in B16F10s assessed by western blot. H, I. and J. Representative western blot analysis (H) and quantification of expression of FADD (I) and FAK (J) in the miR-7a inhibitor or mimic co-transfected with pRK5-FADD experiment in B16F1s assessed by western blot. β-actin served as a loading control. Cells treated with siFADD scramble siRNA negative control (NC) and miR-7a mimic or inhibitor nc served as the sample control in the former experiment, while those treated with pRK5 empty vector plasmid and miR-7a mimic or inhibitor nc served as the sample control in the latter one. K. The systematic schema of the relations between FADD, FAK and miR-7a. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p
Figure Legend Snippet: MiR-7a was a mediator in FADD-regulated expression of FAK A. Firstly, transfected FADD siRNA (siFADD) alone or co-transfected with FADD siRNA (siFADD) and miR-7a mimic or inhibitor into B16F10 cells for 48 hours. Then a 24 h period scratch wound healing assay had been launched in those transfected B16F10 cells after the transfection. Inhibiting miR-7a expression rescued FADD interference reduced cell migration. Migration was retarded even more when miR-7a mimic were used instead of inhibitor co-transfected with FADD siRNAs. B. Firstly transfected pRK5-FADD plasmid alone or co-transfected pRK5-FADD plasmid together with miR-7a mimic or inhibitor into B16F1 cells for 48 hours. Then a 24 h period scratch wound healing assay had been launched in those B16F1 cells after the transfection. FADD overexpression promoted cell migration which is repressible by miR-7a mimic. Cells co-transfected with pRK5-FADD and miR-7a inhibitor exhibited the fastest migration rate among the four different samples. C. and D. B16F1 melanoma cells treated with pRK5 empty vector plasmids, pRK5-FADD plasmids, pRK5-FADD plasmids plus miR-7a mimic and pRK5-FADD plasmids plus miR-7a inhibitor, were collected and suspended in PBS. Then the treated cells were injected into the tail vein of C57BL/6 mice. After 14 days, mice were sacrificed with all the lung tissues were excised and the surface lung tumor nodules were counted. The representative pictures of lung metastases (C) and the number of metastasis foci on the surface of lung (D). Data are presented as mean ± S.D. n=5. E, F. and G. Representative western blot analysis (E) and quantification of expression of FADD (F) and FAK (G) in the miR-7a inhibitor/mimic co-transfected with FADD siRNAs experiment in B16F10s assessed by western blot. H, I. and J. Representative western blot analysis (H) and quantification of expression of FADD (I) and FAK (J) in the miR-7a inhibitor or mimic co-transfected with pRK5-FADD experiment in B16F1s assessed by western blot. β-actin served as a loading control. Cells treated with siFADD scramble siRNA negative control (NC) and miR-7a mimic or inhibitor nc served as the sample control in the former experiment, while those treated with pRK5 empty vector plasmid and miR-7a mimic or inhibitor nc served as the sample control in the latter one. K. The systematic schema of the relations between FADD, FAK and miR-7a. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p

Techniques Used: Expressing, Transfection, Wound Healing Assay, Migration, Plasmid Preparation, Over Expression, Injection, Mouse Assay, Western Blot, Negative Control, Software

FADD regulated FAK expression in MEF cells A. mRNA level of FAK down-regulated in FADD −/− MEF cells. B. and C. Representative western blot analysis (B) and quantification of expression (C) of FADD and FAK in FADD +/+ and −/− MEF cells. α-Tubulin was used as a loading control and the FADD +/+ MEF cell served as the sample control. D. FADD was interfered by 20 μM siRNA (siFADD) in MEF cells for 48 h, scramble siRNAs served as negative control (NC). The mRNA expression of FAK decreased in siFADD samples compared with NC. E. and F. Representative western blot analysis (E) and quantification of expression (F) of FADD and FAK in FADD knockdown MEF cells by siRNA. GAPDH used as a loading control. MEFs treated with scramble siRNAs served as the sample control. G. The cloned pRK5-FADD plasmid was transfected into MEFs to make FADD overexpressed and pRK5 empty vector plasmid as negative control. The mRNA level of FAK increased in cells with pRK5-FADD plasmid compared to the cells with empty plasmid. H. and I. Representative western blot analysis and quantification of expression of FADD and FAK in FADD overexpression MEF cells. GAPDH used as a loading control. MEFs transfected with pRK5 empty vector plasmid acted as the sample control. mRNA levels were monitored by qRT-PCR and proteins were detected with indicated antibodies by western blotting. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p
Figure Legend Snippet: FADD regulated FAK expression in MEF cells A. mRNA level of FAK down-regulated in FADD −/− MEF cells. B. and C. Representative western blot analysis (B) and quantification of expression (C) of FADD and FAK in FADD +/+ and −/− MEF cells. α-Tubulin was used as a loading control and the FADD +/+ MEF cell served as the sample control. D. FADD was interfered by 20 μM siRNA (siFADD) in MEF cells for 48 h, scramble siRNAs served as negative control (NC). The mRNA expression of FAK decreased in siFADD samples compared with NC. E. and F. Representative western blot analysis (E) and quantification of expression (F) of FADD and FAK in FADD knockdown MEF cells by siRNA. GAPDH used as a loading control. MEFs treated with scramble siRNAs served as the sample control. G. The cloned pRK5-FADD plasmid was transfected into MEFs to make FADD overexpressed and pRK5 empty vector plasmid as negative control. The mRNA level of FAK increased in cells with pRK5-FADD plasmid compared to the cells with empty plasmid. H. and I. Representative western blot analysis and quantification of expression of FADD and FAK in FADD overexpression MEF cells. GAPDH used as a loading control. MEFs transfected with pRK5 empty vector plasmid acted as the sample control. mRNA levels were monitored by qRT-PCR and proteins were detected with indicated antibodies by western blotting. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p

Techniques Used: Expressing, Western Blot, Negative Control, Clone Assay, Plasmid Preparation, Transfection, Over Expression, Quantitative RT-PCR, Software

FADD regulated FAK and miR-7a expression in B16F10 and B16F1 melanoma cells A. and B. In highly metastatic B16F10 cells, FADD and FAK exhibited higher level of mRNA (A) and protein (B) expression compared to lowly metastatic B16F1 cells. Conversely, the expression of miR-7a is lower in B16F10 cells than in B16F1. B16F1 cells served as the control sample. C. FADD was interfered by 20 mM siRNA (siFADD) in B16F10 cells for 48 h, scramble siRNAs as negative control (NC). The mRNA expression of FAK decreased in cells with siFADD compared to NC group while the expression of miR-7a increased. D. Representative western blot analysis and quantification of expression of FADD and FAK in FADD knockdown B16F10 cells by siRNA. β-actin used as a loading control. E. FADD was overexpressed by transfected pRK5- FADD plasmid into B16F10 cells for 48 h. The mRNA expression of FAK increased in cells with pRK5-FADD plasmid compared to those with pRK5 empty vector plasmid while miR-7a decreased. F. Representative western blot analysis and quantification of expression of FADD and FAK in FADD overexpressed B16F10 cells. β-actin acted as a loading control. G. and I. The mRNA level of FAK expression also increased while FADD up-regulated (I) and decreased while FADD down-regulated (G) in B16F1 cells. H. and J. Representative western blot analysis and quantification of expression of FADD and FAK in FADD knockdown (H) or overexpressed (J) B16F1 cells. β-actin served as a loading control. Cells treated with siRNA scramble negative control (NC) and pRK5 empty vector plasmid served as the sample control. mRNA levels were detected by qRT-PCR and proteins were detected with indicated antibodies by western blotting. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p
Figure Legend Snippet: FADD regulated FAK and miR-7a expression in B16F10 and B16F1 melanoma cells A. and B. In highly metastatic B16F10 cells, FADD and FAK exhibited higher level of mRNA (A) and protein (B) expression compared to lowly metastatic B16F1 cells. Conversely, the expression of miR-7a is lower in B16F10 cells than in B16F1. B16F1 cells served as the control sample. C. FADD was interfered by 20 mM siRNA (siFADD) in B16F10 cells for 48 h, scramble siRNAs as negative control (NC). The mRNA expression of FAK decreased in cells with siFADD compared to NC group while the expression of miR-7a increased. D. Representative western blot analysis and quantification of expression of FADD and FAK in FADD knockdown B16F10 cells by siRNA. β-actin used as a loading control. E. FADD was overexpressed by transfected pRK5- FADD plasmid into B16F10 cells for 48 h. The mRNA expression of FAK increased in cells with pRK5-FADD plasmid compared to those with pRK5 empty vector plasmid while miR-7a decreased. F. Representative western blot analysis and quantification of expression of FADD and FAK in FADD overexpressed B16F10 cells. β-actin acted as a loading control. G. and I. The mRNA level of FAK expression also increased while FADD up-regulated (I) and decreased while FADD down-regulated (G) in B16F1 cells. H. and J. Representative western blot analysis and quantification of expression of FADD and FAK in FADD knockdown (H) or overexpressed (J) B16F1 cells. β-actin served as a loading control. Cells treated with siRNA scramble negative control (NC) and pRK5 empty vector plasmid served as the sample control. mRNA levels were detected by qRT-PCR and proteins were detected with indicated antibodies by western blotting. Band intensity was quantified by Image J software. The results shown are representative of three different experiments. Data are represented as mean ± S.D. *p

Techniques Used: Expressing, Negative Control, Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, Software

FADD and FAK overexpression was associated with human melanoma cancer progression A. Oncomine analysis of FADD expression in human Melanoma cancer. B. Oncomine analysis of FAK expression in human Melanoma cancer. C. Correlation analysis of FADD and FAK expression in human normal skin. D. Correlation analysis of FADD and FAK expression in human melanoma cancer.
Figure Legend Snippet: FADD and FAK overexpression was associated with human melanoma cancer progression A. Oncomine analysis of FADD expression in human Melanoma cancer. B. Oncomine analysis of FAK expression in human Melanoma cancer. C. Correlation analysis of FADD and FAK expression in human normal skin. D. Correlation analysis of FADD and FAK expression in human melanoma cancer.

Techniques Used: Over Expression, Expressing

17) Product Images from "Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, β1-integrin, and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts"

Article Title: Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, β1-integrin, and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22020942

Extracellular PEPD induced expression of the β 1 -integrin receptor and IGF-1R signaling proteins in control and “scratched” fibroblast models. ( A ) The proteins of β 1 -integrin receptor and IGF-1R downstream signaling pathways, FAK, Grb2, and ( C ) NF-ĸβ and ERK1/2 were analyzed by Western blot in lysates of PEPD-treated fibroblasts (50, and 100 nM). Representative blot images were shown (densitometry of protein stains is presented under protein bands as a ratio versus control; Supplementary Figure S2 ). GAPDH was used as a loading control. ( B ) Illustration of the β 1 -integrin receptor-downstream signaling pathway. Created with BioRender.com.
Figure Legend Snippet: Extracellular PEPD induced expression of the β 1 -integrin receptor and IGF-1R signaling proteins in control and “scratched” fibroblast models. ( A ) The proteins of β 1 -integrin receptor and IGF-1R downstream signaling pathways, FAK, Grb2, and ( C ) NF-ĸβ and ERK1/2 were analyzed by Western blot in lysates of PEPD-treated fibroblasts (50, and 100 nM). Representative blot images were shown (densitometry of protein stains is presented under protein bands as a ratio versus control; Supplementary Figure S2 ). GAPDH was used as a loading control. ( B ) Illustration of the β 1 -integrin receptor-downstream signaling pathway. Created with BioRender.com.

Techniques Used: Expressing, Western Blot

18) Product Images from "Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin \u03b21 to Modulate Malignant Properties of Hepatoma Cells"

Article Title: Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin \u03b21 to Modulate Malignant Properties of Hepatoma Cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.277699

Activation of downstream FAK signaling pathway induced by HAb18G/CD147-integrin β1 subunit interaction. A , design of the shRNA. Top , structure of small hairpin RNAs targeting CD147 mRNA. Middle , structure of small hairpin RNAs acting as a negative
Figure Legend Snippet: Activation of downstream FAK signaling pathway induced by HAb18G/CD147-integrin β1 subunit interaction. A , design of the shRNA. Top , structure of small hairpin RNAs targeting CD147 mRNA. Middle , structure of small hairpin RNAs acting as a negative

Techniques Used: Activation Assay, shRNA

19) Product Images from "The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells"

Article Title: The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells

Journal: Oncogene

doi: 10.1038/onc.2008.383

MSP-stimulation of RON leads to MAPK and AKT activation (a)Cell lysates were prepared after 3 hours of serum starvation and 30 minute stimulation with 200ng/ml MSP. Whole cell lysates were run on a polyacrylamide gel and subjected to blotting with phospho-MAPK, MAPK, phospho-AKT and AKT antibodies. (b) Focal adhesion kinase was immunoprecipitated from cells and subjected to a Western Blot analysis by 4G10 phospho-tyrosine antibodies. The blot was stripped and reprobed for total FAK expression.
Figure Legend Snippet: MSP-stimulation of RON leads to MAPK and AKT activation (a)Cell lysates were prepared after 3 hours of serum starvation and 30 minute stimulation with 200ng/ml MSP. Whole cell lysates were run on a polyacrylamide gel and subjected to blotting with phospho-MAPK, MAPK, phospho-AKT and AKT antibodies. (b) Focal adhesion kinase was immunoprecipitated from cells and subjected to a Western Blot analysis by 4G10 phospho-tyrosine antibodies. The blot was stripped and reprobed for total FAK expression.

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Expressing

Related Articles

Protein Binding:

Article Title: Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model
Article Snippet: .. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis–Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). .. After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA).

Immunodetection:

Article Title: Intracellular role of IL-6 in mesenchymal stromal cell immunosuppression and proliferation
Article Snippet: .. Immunodetection was performed with the following primary antibodies: IL-6, (rabbit), cyclin D1 (mouse) (both from Santa Cruz Biotechnology, Sant Cruz, CA), ERK1/2, phospho-ERK1/2 (BD biosciences, Mouse) and GAPDH (Sigma-Aldrich, Mouse). .. Secondary antibodies were HRP-conjugated sheep anti-rabbit IgG or HRP-conjugated sheep anti-mouse IgG (GE Healthcare Amersham, Little Chalfont, UK).

Blocking Assay:

Article Title: Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model
Article Snippet: .. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis–Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). .. After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA).

Electrophoresis:

Article Title: Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model
Article Snippet: .. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis–Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). .. After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA).

Incubation:

Article Title: Fusobacterium nucleatum Accelerates the Progression of Colitis-Associated Colorectal Cancer by Promoting EMT
Article Snippet: .. The membranes were incubated overnight at 4 °C with the primary antibodies against E-cadherin (Cell Signaling, #3195, Danvers, MA, USA), fibronectin (BD Bioscience, #610077 San Jose, CA, USA and Santa Cruz, #SC-9068, CA, USA), Snail (Cell Signaling, #3879, Danvers, MA, USA), p-EGFR (Invitrogen, #18-2463, Carlsbad, CA, USA), EGFR (BD Bioscience, Santa Cruz, #SC-03, CA, USA), p-AKT (Cell Signaling, #9271, Danvers, MA, USA), AKT (Cell Signaling, #9272, Danvers, MA, USA), p-ERK1/2 (BD Bioscience, Santa Cruz, #SC-7976, CA, USA), ERK1/2 (BD Biosciences, Santa Cruz, #SC-94, CA, USA), and β-actin (BD Bioscience, Santa Cruz, #SC-47778, CA, USA). ..

Article Title: FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway
Article Snippet: .. Immunoblots were incubated overnight at 4 °C with primary antibodies including anti-NaPi-2a (generous gift of Drs. Jürg Biber and Heini Murer, University of Zurich), anti-total-ERK1/2 (BD Biosciences), anti-phospho-ERK1/2 (Cell Signaling), anti-total-SGK1 (Applied Biosystems), anti-phospho-SGK1 (Santa Cruz Biotechnology), anti-αKlotho (Alpha Diagnostics, 1:1000), or anti-β-actin (Sigma) antibody in 2% (w/v) bovine serum albumin (BSA, Sigma) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. .. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Life Sciences).

Article Title: Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model
Article Snippet: .. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis–Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). .. After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA).

Western Blot:

Article Title: FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway
Article Snippet: .. Immunoblots were incubated overnight at 4 °C with primary antibodies including anti-NaPi-2a (generous gift of Drs. Jürg Biber and Heini Murer, University of Zurich), anti-total-ERK1/2 (BD Biosciences), anti-phospho-ERK1/2 (Cell Signaling), anti-total-SGK1 (Applied Biosystems), anti-phospho-SGK1 (Santa Cruz Biotechnology), anti-αKlotho (Alpha Diagnostics, 1:1000), or anti-β-actin (Sigma) antibody in 2% (w/v) bovine serum albumin (BSA, Sigma) in a TBS-T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. .. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Life Sciences).

Acrylamide Gel Assay:

Article Title: Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model
Article Snippet: .. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis–Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). .. After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA).

Staining:

Article Title: Loss of DEK induces radioresistance of murine restricted hematopoietic progenitors
Article Snippet: .. After washing, cells were stained intracellularly using Alexa Fluor® -647 anti-phospho-p38(clone 36/p38 (pT180/pY182), BD Biosciences) and Alexa Fluor® -647 anti-phospho-ERK1/2 (clone 20A, BD Biosciences) for 40 minutes in Perm/Wash Buffer 1× (BD Bioscience) with 0.5% of mouse serum. ..

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  • 94
    Becton Dickinson anti fak mab
    Model depicting the intracellular events during <t>integrin</t> α 2 β 1 –mediated platelet spreading. Integrin α 2 β 1 mediates activation of a Src family kinase, Syk, and SLP-76 upstream of PLCγ2, Ca 2+ mobilization, and platelet spreading. A second pathway can also give rise to Ca 2+ mobilization and platelet spreading. This pathway may involve the tyrosine kinase <t>FAK,</t> whose phosphorylation is mediated, in part, through a Src kinase–independent pathway. A possible pathway of FAK-mediated increase in calcium is the inhibition of PMCA via tyrosine phosphorylation.
    Anti Fak Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti fak
    Involvement of ERK, <t>FAK,</t> and PI3K/Akt pathways in upregulation of MMP-2 driven by soluble CD147. SMMC-7721 cells were cultured for 30 min (A) and 12 h (B) with 10 μg/ml of E-CD147ECD. CD147 and MMP-2 data were expressed as means ± SEM of three independent determinations of CD147 or MMP-2/α-tubulin ratio. (C) SMMC-7721 cells were treated with E-CD147ECD (10 μg/ml) for 12 h with or without pre-treatment with ERK inhibitor (U0126), FAK inhibitor (FAK inhibitor 14), PI3K inhibitor (LY294002), or combined inhibitors. In A and C, the expression levels of <t>phospho-ERK1/2,</t> ERK1/2, phospho-FAK, FAK, phospho-Akt, Akt, phospho-EGFR, and EGFR were detected by western blotting. Quantitative analysis of phosphorylated fraction relative to the total fraction was shown. Mean values for control groups were normalized to 1. * P
    Anti Fak, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Becton Dickinson anti fak antibody
    The MOA study of RSV inhibiting cell migration. The data are represented as the mean ± SEM. (A) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. <t>FAK</t> expression in the cells was measured with qPCR. (B) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. FAK expression in the cells was measured with western blotting. (C) B16F10 cells with or without the transfection of the plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. FAK expression in the cells was measured with western blotting. (D) B16F10 cells with or without the transfection of plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. Cell migration rate was measured with RTCA assays. (E) B16F10 cells were transfected with the FAK promoter reporter gene plasmid followed by incubation with different concentrations of RSV for 24 h. The FAK promoter activity was measured with dual-luciferase reporter assays. (F) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and <t>DNMT3a</t> for 24 h. The binding activity of HDAC1 and DNMT3a with the FAK promoter was measured with ChIP assays. (G) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The methylation level of the FAK promoter in the cells was measured with BSP assays. (H) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The binding activity of USF1 with the FAK promoter was measured with ChIP assays. (I) The methylation conditions of the CpG sites near position -40 of the FAK promoter following treatments with RSV and its analogs. (J) B16F10 cells were treated with RSV and the inhibitors of HDAC1 and DNMT3a for 24 h. The cell migration rates were measured with RTCA assays. The concentrations of DAC and TSA were 200 ng/mL and 40 nM, respectively.
    Anti Fak Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 96/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Model depicting the intracellular events during integrin α 2 β 1 –mediated platelet spreading. Integrin α 2 β 1 mediates activation of a Src family kinase, Syk, and SLP-76 upstream of PLCγ2, Ca 2+ mobilization, and platelet spreading. A second pathway can also give rise to Ca 2+ mobilization and platelet spreading. This pathway may involve the tyrosine kinase FAK, whose phosphorylation is mediated, in part, through a Src kinase–independent pathway. A possible pathway of FAK-mediated increase in calcium is the inhibition of PMCA via tyrosine phosphorylation.

    Journal: The Journal of Cell Biology

    Article Title: Integrin ?2?1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLC?2

    doi: 10.1083/jcb.200208043

    Figure Lengend Snippet: Model depicting the intracellular events during integrin α 2 β 1 –mediated platelet spreading. Integrin α 2 β 1 mediates activation of a Src family kinase, Syk, and SLP-76 upstream of PLCγ2, Ca 2+ mobilization, and platelet spreading. A second pathway can also give rise to Ca 2+ mobilization and platelet spreading. This pathway may involve the tyrosine kinase FAK, whose phosphorylation is mediated, in part, through a Src kinase–independent pathway. A possible pathway of FAK-mediated increase in calcium is the inhibition of PMCA via tyrosine phosphorylation.

    Article Snippet: Purified anti–mouse α2 integrin mAb (HMα2), hamster IgG1κ (A19-3), anti–SLP-76 mAb, and anti-FAK mAb (clone 8) for Western blotting were from Becton Dickinson.

    Techniques: Activation Assay, Inhibition

    Integrin α 2 β 1 –mediated platelet spreading induces protein tyrosine phosphorylation. (A) Washed human platelets were pretreated with 100 μM EGTA, 10 μM indomethacin, 10 μM lotrafiban, and 3 U/ml apyrase. Where indicated, 10 μM ADP was added and apyrase omitted. Platelets were pretreated with 20 μM PP2 as shown. For adhesion studies, 500 μl washed platelets (4 × 10 8 /ml) was seeded on the dishes coated with 1% BSA (C, control) or 50 μg/ml GFOGER at 30°C for the indicated times. After removal of unbound platelets, adhered platelets were dissolved by 500 μl Laemmli sample buffer. Unbound platelets that had been exposed to a BSA-coated surface served as the zero time point. For suspension studies, 500 μl washed platelets (4 × 10 8 /ml) in a glass cuvette was stimulated with 10 μM ADP and then incubated with or without 50 μg/ml GFOGER for the indicated times with stirring at 30°C. After the protein concentrations of each sample were adjusted, proteins were separated by 8% SDS-PAGE, and protein tyrosine phosphorylation was blotted with 4G10. Results are representative of two separate experiments. (B) Washed human platelets (3 × 10 8 /ml) were pretreated with the inhibitors and stimulated as described above. Reactions were stopped by the addition of an equal volume of 2× lysis buffer without removing unbound platelets. FAK (1) or PLCγ2 (2) proteins were isolated by immunoprecipitation with anti-FAK pAb or anti-PLCγ2 pAb and blotted with 4G10, anti-FAK mAb, or anti-PLCγ2 pAb. Results are representative of three separate experiments. (C) Washed human platelets (3 × 10 8 /ml) were pretreated with 100 μM EGTA, 10 μM indomethacin, 10 μM lotrafiban, and 10 μM ADP. 20 μM PP2 was added as shown. Reactions were stopped, and FAK (1) or PLCγ2 (2) proteins were isolated by immunoprecipitation as described above. Results are representative of three separate experiments.

    Journal: The Journal of Cell Biology

    Article Title: Integrin ?2?1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLC?2

    doi: 10.1083/jcb.200208043

    Figure Lengend Snippet: Integrin α 2 β 1 –mediated platelet spreading induces protein tyrosine phosphorylation. (A) Washed human platelets were pretreated with 100 μM EGTA, 10 μM indomethacin, 10 μM lotrafiban, and 3 U/ml apyrase. Where indicated, 10 μM ADP was added and apyrase omitted. Platelets were pretreated with 20 μM PP2 as shown. For adhesion studies, 500 μl washed platelets (4 × 10 8 /ml) was seeded on the dishes coated with 1% BSA (C, control) or 50 μg/ml GFOGER at 30°C for the indicated times. After removal of unbound platelets, adhered platelets were dissolved by 500 μl Laemmli sample buffer. Unbound platelets that had been exposed to a BSA-coated surface served as the zero time point. For suspension studies, 500 μl washed platelets (4 × 10 8 /ml) in a glass cuvette was stimulated with 10 μM ADP and then incubated with or without 50 μg/ml GFOGER for the indicated times with stirring at 30°C. After the protein concentrations of each sample were adjusted, proteins were separated by 8% SDS-PAGE, and protein tyrosine phosphorylation was blotted with 4G10. Results are representative of two separate experiments. (B) Washed human platelets (3 × 10 8 /ml) were pretreated with the inhibitors and stimulated as described above. Reactions were stopped by the addition of an equal volume of 2× lysis buffer without removing unbound platelets. FAK (1) or PLCγ2 (2) proteins were isolated by immunoprecipitation with anti-FAK pAb or anti-PLCγ2 pAb and blotted with 4G10, anti-FAK mAb, or anti-PLCγ2 pAb. Results are representative of three separate experiments. (C) Washed human platelets (3 × 10 8 /ml) were pretreated with 100 μM EGTA, 10 μM indomethacin, 10 μM lotrafiban, and 10 μM ADP. 20 μM PP2 was added as shown. Reactions were stopped, and FAK (1) or PLCγ2 (2) proteins were isolated by immunoprecipitation as described above. Results are representative of three separate experiments.

    Article Snippet: Purified anti–mouse α2 integrin mAb (HMα2), hamster IgG1κ (A19-3), anti–SLP-76 mAb, and anti-FAK mAb (clone 8) for Western blotting were from Becton Dickinson.

    Techniques: Incubation, SDS Page, Lysis, Isolation, Immunoprecipitation

    Involvement of ERK, FAK, and PI3K/Akt pathways in upregulation of MMP-2 driven by soluble CD147. SMMC-7721 cells were cultured for 30 min (A) and 12 h (B) with 10 μg/ml of E-CD147ECD. CD147 and MMP-2 data were expressed as means ± SEM of three independent determinations of CD147 or MMP-2/α-tubulin ratio. (C) SMMC-7721 cells were treated with E-CD147ECD (10 μg/ml) for 12 h with or without pre-treatment with ERK inhibitor (U0126), FAK inhibitor (FAK inhibitor 14), PI3K inhibitor (LY294002), or combined inhibitors. In A and C, the expression levels of phospho-ERK1/2, ERK1/2, phospho-FAK, FAK, phospho-Akt, Akt, phospho-EGFR, and EGFR were detected by western blotting. Quantitative analysis of phosphorylated fraction relative to the total fraction was shown. Mean values for control groups were normalized to 1. * P

    Journal: Journal of Translational Medicine

    Article Title: Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma

    doi: 10.1186/1479-5876-12-190

    Figure Lengend Snippet: Involvement of ERK, FAK, and PI3K/Akt pathways in upregulation of MMP-2 driven by soluble CD147. SMMC-7721 cells were cultured for 30 min (A) and 12 h (B) with 10 μg/ml of E-CD147ECD. CD147 and MMP-2 data were expressed as means ± SEM of three independent determinations of CD147 or MMP-2/α-tubulin ratio. (C) SMMC-7721 cells were treated with E-CD147ECD (10 μg/ml) for 12 h with or without pre-treatment with ERK inhibitor (U0126), FAK inhibitor (FAK inhibitor 14), PI3K inhibitor (LY294002), or combined inhibitors. In A and C, the expression levels of phospho-ERK1/2, ERK1/2, phospho-FAK, FAK, phospho-Akt, Akt, phospho-EGFR, and EGFR were detected by western blotting. Quantitative analysis of phosphorylated fraction relative to the total fraction was shown. Mean values for control groups were normalized to 1. * P

    Article Snippet: The membrane was probed with primary antibodies including HAb18, C-19 (Santa Cruz Biotechnology), anti-MMP-2 (Santa Cruz Biotechnology), anti-p-ERK1/2, anti-p-FAK, anti-p-Akt, anti-p-EGFR, anti-ERK1/2, anti-Akt, anti-EGFR (Cell Signaling Technology, Danvers, USA), anti-FAK (BD), and anti-α-tubulin antibodies (Santa Cruz Biotechnology).

    Techniques: Cell Culture, Expressing, Western Blot

    The MOA study of RSV inhibiting cell migration. The data are represented as the mean ± SEM. (A) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. FAK expression in the cells was measured with qPCR. (B) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. FAK expression in the cells was measured with western blotting. (C) B16F10 cells with or without the transfection of the plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. FAK expression in the cells was measured with western blotting. (D) B16F10 cells with or without the transfection of plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. Cell migration rate was measured with RTCA assays. (E) B16F10 cells were transfected with the FAK promoter reporter gene plasmid followed by incubation with different concentrations of RSV for 24 h. The FAK promoter activity was measured with dual-luciferase reporter assays. (F) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The binding activity of HDAC1 and DNMT3a with the FAK promoter was measured with ChIP assays. (G) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The methylation level of the FAK promoter in the cells was measured with BSP assays. (H) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The binding activity of USF1 with the FAK promoter was measured with ChIP assays. (I) The methylation conditions of the CpG sites near position -40 of the FAK promoter following treatments with RSV and its analogs. (J) B16F10 cells were treated with RSV and the inhibitors of HDAC1 and DNMT3a for 24 h. The cell migration rates were measured with RTCA assays. The concentrations of DAC and TSA were 200 ng/mL and 40 nM, respectively.

    Journal: Theranostics

    Article Title: Comparative profiling of analog targets: a case study on resveratrol for mouse melanoma metastasis suppression

    doi: 10.7150/thno.24336

    Figure Lengend Snippet: The MOA study of RSV inhibiting cell migration. The data are represented as the mean ± SEM. (A) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. FAK expression in the cells was measured with qPCR. (B) B16F10 cells were treated with different concentrations of RSV and the analogs for 24 h. FAK expression in the cells was measured with western blotting. (C) B16F10 cells with or without the transfection of the plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. FAK expression in the cells was measured with western blotting. (D) B16F10 cells with or without the transfection of plasmid that overexpresses FAK were treated with 60 μM RSV for 24 h. Cell migration rate was measured with RTCA assays. (E) B16F10 cells were transfected with the FAK promoter reporter gene plasmid followed by incubation with different concentrations of RSV for 24 h. The FAK promoter activity was measured with dual-luciferase reporter assays. (F) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The binding activity of HDAC1 and DNMT3a with the FAK promoter was measured with ChIP assays. (G) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The methylation level of the FAK promoter in the cells was measured with BSP assays. (H) B16F10 cells were treated with RSV, its analogs and the inhibitors of HDAC1 and DNMT3a for 24 h. The binding activity of USF1 with the FAK promoter was measured with ChIP assays. (I) The methylation conditions of the CpG sites near position -40 of the FAK promoter following treatments with RSV and its analogs. (J) B16F10 cells were treated with RSV and the inhibitors of HDAC1 and DNMT3a for 24 h. The cell migration rates were measured with RTCA assays. The concentrations of DAC and TSA were 200 ng/mL and 40 nM, respectively.

    Article Snippet: The antibodies used in the study were anti-DNMT3a antibody (Abcam; ab13888), anti-HDAC1 antibody (Abcam; ab7028), anti-ACAT1 antibody (Abcam; ab168342), anti-FAK antibody (BD; 610088), anti-β-actin antibody (CST; 12262), anti-AMPKα antibody (CST; 2532), and anti-phospho-AMPKα antibody (CST; 2535).

    Techniques: Migration, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Incubation, Activity Assay, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Methylation