phosph fak Search Results


antihuman phosphorylated fak antibody  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology antihuman phosphorylated fak antibody
Fig. 4. Egfl7 facilitates <t>FAK</t> phosphorylation and promotes motility of HCCLM3Egfl7RNAi cells. (A) Western blot results showed that the <t>phosphorylated</t> FAK level in HCCLM3Egfl7RNAi cells was significantly lower than that in HCCLM3Egfl7RNAi cells (P 0.01), but the total FAK levels in these cells were close (P 0.05). (B) When HCCLM3Egfl7RNAi cells were treated by recombinant Egfl7 protein (50 ng/mL), the phosphorylated FAK level was elevated significantly (P 0.01) but the total FAK level did not change (P 0.05). (C) For immunofluorescence studies, HCCLM3Egfl7RNAi cells growing on a coverslip in 6-well plates were kept on serum-free media for 24 hours before it was treated with recombinant Egfl7 protein (50 ng/mL) for 60 minutes at 37°C. After cells were fixed in 3.7% formaldehyde solution, F-actin filaments were visualized in cells using rhodamine-conjugated phalloidin. (D) The wound healing assay showed that the migration of HCCLM3Egfl7RNAi cells was enhanced after recombinant Egfl7 protein (50 ng/mL) treatment (74% versus 49%, P 0.05). (E) The Transwell assay showed that the number of HCCLM3Egfl7RNAi that traveled through Matrigel cells was significantly elevated after recombinant Egfl7 protein (50 ng/mL) treatment (108 27 versus 48 9, P 0.05). Western blot, immunofluorescence, wound healing assay, and Transwell assay were all performed in triplicate. The abundance of FAK and phosphorylated FAK protein in cells are shown relative to those of -actin protein. **P 0.01; *P 0.05.
Antihuman Phosphorylated Fak Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pfak tyr925  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc pfak tyr925
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Pfak Tyr925, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-/total paxillin antibody  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc phospho-/total paxillin antibody
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Phospho /Total Paxillin Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti rabbit monoclonal  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc anti rabbit monoclonal
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Anti Rabbit Monoclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti pfak tyr 576 577 antibody  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc anti pfak tyr 576 577 antibody
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Anti Pfak Tyr 576 577 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-phospho-fak tyr-576-r (rabbit pab)  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology anti-phospho-fak tyr-576-r (rabbit pab)
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Anti Phospho Fak Tyr 576 R (Rabbit Pab), supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho fak py397  (Thermo Fisher)
86
Thermo Fisher phospho fak py397
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Phospho Fak Py397, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-y397 fak  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc phospho-y397 fak
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Phospho Y397 Fak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit polyclonal anti fak  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit polyclonal anti fak
Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK <t>(pFAK,</t> Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.
Rabbit Polyclonal Anti Fak, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti pfak y397  (Thermo Fisher)
86
Thermo Fisher rabbit anti pfak y397
LIMCH1 depletion affects the formation of focal adhesions in the cell center and phosphorylation of FAK. (A) Binary images of focal adhesions stained with anti-paxillin antibody in siRNA-treated HeLa cells were processed using ImageJ software. Cell outlines (red) and central areas (green) were defined by staining with phalloidin and NM-IIB, respectively. (B) Quantification of the number of total focal adhesions. (C) The ratio of focal adhesions was measured by dividing the number of central or peripheral adhesions by the number of total focal adhesion. In B and C, results were analyzed using ImageJ software ( n = 118–142 cells in three independent experiments, mean ± SD, * p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). (D) Cell extracts from siRNA-treated cells were probed with <t>anti-pFAK</t> <t>Y397</t> and FAK antibodies. (E) Relative levels of pFAK Y397 shown in D ( n = 4, mean ± SD, normalized to siRNA control, ** p < 0.001, two-tailed t test).
Rabbit Anti Pfak Y397, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-fak  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology phospho-fak
LIMCH1 depletion affects the formation of focal adhesions in the cell center and phosphorylation of FAK. (A) Binary images of focal adhesions stained with anti-paxillin antibody in siRNA-treated HeLa cells were processed using ImageJ software. Cell outlines (red) and central areas (green) were defined by staining with phalloidin and NM-IIB, respectively. (B) Quantification of the number of total focal adhesions. (C) The ratio of focal adhesions was measured by dividing the number of central or peripheral adhesions by the number of total focal adhesion. In B and C, results were analyzed using ImageJ software ( n = 118–142 cells in three independent experiments, mean ± SD, * p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). (D) Cell extracts from siRNA-treated cells were probed with <t>anti-pFAK</t> <t>Y397</t> and FAK antibodies. (E) Relative levels of pFAK Y397 shown in D ( n = 4, mean ± SD, normalized to siRNA control, ** p < 0.001, two-tailed t test).
Phospho Fak, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-fak  (Becton Dickinson)
90
Becton Dickinson phospho-fak
LIMCH1 depletion affects the formation of focal adhesions in the cell center and phosphorylation of FAK. (A) Binary images of focal adhesions stained with anti-paxillin antibody in siRNA-treated HeLa cells were processed using ImageJ software. Cell outlines (red) and central areas (green) were defined by staining with phalloidin and NM-IIB, respectively. (B) Quantification of the number of total focal adhesions. (C) The ratio of focal adhesions was measured by dividing the number of central or peripheral adhesions by the number of total focal adhesion. In B and C, results were analyzed using ImageJ software ( n = 118–142 cells in three independent experiments, mean ± SD, * p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). (D) Cell extracts from siRNA-treated cells were probed with <t>anti-pFAK</t> <t>Y397</t> and FAK antibodies. (E) Relative levels of pFAK Y397 shown in D ( n = 4, mean ± SD, normalized to siRNA control, ** p < 0.001, two-tailed t test).
Phospho Fak, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Fig. 4. Egfl7 facilitates FAK phosphorylation and promotes motility of HCCLM3Egfl7RNAi cells. (A) Western blot results showed that the phosphorylated FAK level in HCCLM3Egfl7RNAi cells was significantly lower than that in HCCLM3Egfl7RNAi cells (P 0.01), but the total FAK levels in these cells were close (P 0.05). (B) When HCCLM3Egfl7RNAi cells were treated by recombinant Egfl7 protein (50 ng/mL), the phosphorylated FAK level was elevated significantly (P 0.01) but the total FAK level did not change (P 0.05). (C) For immunofluorescence studies, HCCLM3Egfl7RNAi cells growing on a coverslip in 6-well plates were kept on serum-free media for 24 hours before it was treated with recombinant Egfl7 protein (50 ng/mL) for 60 minutes at 37°C. After cells were fixed in 3.7% formaldehyde solution, F-actin filaments were visualized in cells using rhodamine-conjugated phalloidin. (D) The wound healing assay showed that the migration of HCCLM3Egfl7RNAi cells was enhanced after recombinant Egfl7 protein (50 ng/mL) treatment (74% versus 49%, P 0.05). (E) The Transwell assay showed that the number of HCCLM3Egfl7RNAi that traveled through Matrigel cells was significantly elevated after recombinant Egfl7 protein (50 ng/mL) treatment (108 27 versus 48 9, P 0.05). Western blot, immunofluorescence, wound healing assay, and Transwell assay were all performed in triplicate. The abundance of FAK and phosphorylated FAK protein in cells are shown relative to those of -actin protein. **P 0.01; *P 0.05.

Journal: Hepatology (Baltimore, Md.)

Article Title: Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

doi: 10.1002/hep.23197

Figure Lengend Snippet: Fig. 4. Egfl7 facilitates FAK phosphorylation and promotes motility of HCCLM3Egfl7RNAi cells. (A) Western blot results showed that the phosphorylated FAK level in HCCLM3Egfl7RNAi cells was significantly lower than that in HCCLM3Egfl7RNAi cells (P 0.01), but the total FAK levels in these cells were close (P 0.05). (B) When HCCLM3Egfl7RNAi cells were treated by recombinant Egfl7 protein (50 ng/mL), the phosphorylated FAK level was elevated significantly (P 0.01) but the total FAK level did not change (P 0.05). (C) For immunofluorescence studies, HCCLM3Egfl7RNAi cells growing on a coverslip in 6-well plates were kept on serum-free media for 24 hours before it was treated with recombinant Egfl7 protein (50 ng/mL) for 60 minutes at 37°C. After cells were fixed in 3.7% formaldehyde solution, F-actin filaments were visualized in cells using rhodamine-conjugated phalloidin. (D) The wound healing assay showed that the migration of HCCLM3Egfl7RNAi cells was enhanced after recombinant Egfl7 protein (50 ng/mL) treatment (74% versus 49%, P 0.05). (E) The Transwell assay showed that the number of HCCLM3Egfl7RNAi that traveled through Matrigel cells was significantly elevated after recombinant Egfl7 protein (50 ng/mL) treatment (108 27 versus 48 9, P 0.05). Western blot, immunofluorescence, wound healing assay, and Transwell assay were all performed in triplicate. The abundance of FAK and phosphorylated FAK protein in cells are shown relative to those of -actin protein. **P 0.01; *P 0.05.

Article Snippet: The blotted membranes were incubated antihuman Egfl7 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), antihuman FAK antibody (Santa Cruz), or antihuman phosphorylated FAK antibody (Santa Cruz) and then secondary antibody (KPL, Gaithersburg, MD) in order.

Techniques: Phospho-proteomics, Western Blot, Recombinant, Wound Healing Assay, Migration, Transwell Assay

Fig. 5. EGFR inhibitor blocks the effect of Egfl7 on FAK phosphorylation and cell motility. (A) Western blot results showed that the phosphorylated FAK level in HCCLM3Egfl7RNAi cells treated by EGFR inhibitor (15 M) was significantly lower than that in HCCLM3Egfl7RNAi cells treated by PBS after stimulation of the recombinant Egfl7 protein (50 ng/mL) (P 0.01). (B) Immunofluorescence data showed that the EGFR inhibitor (15 M) blocked the reorganization of actin in HCCLM3Egfl7RNAi cells induced by recombinant Egfl7 protein (50 ng/mL). (C) The wound healing assay showed that the migration of HCCLM3Egfl7RNAi cells induced by the recombinant Egfl7 protein (50 ng/mL) was inhibited by EGFR inhibitor (15 M) treatment (27% versus 73%, P 0.05). (D) The Transwell assay also showed that number of HCCLM3Egfl7RNAi cells invaded through Matrigel cells was significantly decreased under EGFR inhibitor (15 M) treatment after recombinant Egfl7 (50 ng/mL) protein stimulation (49 5 versus 107 17, P 0.05). Western blot, immunofluorescence, wound healing assay, and Transwell assay were all performed in triplicate. The abundance of FAK and phosphorylated FAK protein in cells are shown relative to those of -actin protein. **P 0.01; *P 0.05.

Journal: Hepatology (Baltimore, Md.)

Article Title: Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

doi: 10.1002/hep.23197

Figure Lengend Snippet: Fig. 5. EGFR inhibitor blocks the effect of Egfl7 on FAK phosphorylation and cell motility. (A) Western blot results showed that the phosphorylated FAK level in HCCLM3Egfl7RNAi cells treated by EGFR inhibitor (15 M) was significantly lower than that in HCCLM3Egfl7RNAi cells treated by PBS after stimulation of the recombinant Egfl7 protein (50 ng/mL) (P 0.01). (B) Immunofluorescence data showed that the EGFR inhibitor (15 M) blocked the reorganization of actin in HCCLM3Egfl7RNAi cells induced by recombinant Egfl7 protein (50 ng/mL). (C) The wound healing assay showed that the migration of HCCLM3Egfl7RNAi cells induced by the recombinant Egfl7 protein (50 ng/mL) was inhibited by EGFR inhibitor (15 M) treatment (27% versus 73%, P 0.05). (D) The Transwell assay also showed that number of HCCLM3Egfl7RNAi cells invaded through Matrigel cells was significantly decreased under EGFR inhibitor (15 M) treatment after recombinant Egfl7 (50 ng/mL) protein stimulation (49 5 versus 107 17, P 0.05). Western blot, immunofluorescence, wound healing assay, and Transwell assay were all performed in triplicate. The abundance of FAK and phosphorylated FAK protein in cells are shown relative to those of -actin protein. **P 0.01; *P 0.05.

Article Snippet: The blotted membranes were incubated antihuman Egfl7 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), antihuman FAK antibody (Santa Cruz), or antihuman phosphorylated FAK antibody (Santa Cruz) and then secondary antibody (KPL, Gaithersburg, MD) in order.

Techniques: Phospho-proteomics, Western Blot, Recombinant, Wound Healing Assay, Migration, Transwell Assay

Fig. 6. Knockdown of Egfl7 inhibits the growth and metastasis of HCCLM3 cells in vivo. (A) The size of liver tumors in the HCCLM3Egfl7RNAi group was dramatically smaller than that of the HCCLM3Egfl7RNAi group. Black arrows: local tumors at the sites of implantation. The volume of liver tumors was calculated as follows: tumor volume (mm3) length width thickness. Relative tumor volume (volume of tumor tissue)/(volume of initially planted tumor tissue). (B) H&E staining was used in the HCCLM3Egfl7RNAi group (a) and the HCCLM3Egfl7RNAi group (b) and immunohistochemistry examination for Egfl7 and phosphorylated FAK expression in tumor was also performed in the HCCLM3Egfl7RNAi group (c,e) and the HCCLM3Egfl7RNAi group (d,f). Original magnification 400. (C) Metastasis of HCCLM3: (a) intrahepatic metastasis of HCCLM3Egfl7RNAi group (yellow arrow, local tumor; green arrows, metastatic nodules); (b) pulmonary metastasis of HCCLM3Egfl7RNAi group (blue arrow, metastatic HCCLM3 cells in the lung tissues of the mice, H&E stain, original magnification 400). (D) MVD was calculated in tumors of the HCCLM3Egfl7RNAi group (a) and HCCLM3Egfl7RNAi group (b) and the results showed that the average MVD in tumors of the HCCLM3Egfl7RNAi group was significantly lower than that in the HCCLM3Egfl7RNAi group (24 11 versus 49 23, P 0.05). *P 0.05.

Journal: Hepatology (Baltimore, Md.)

Article Title: Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

doi: 10.1002/hep.23197

Figure Lengend Snippet: Fig. 6. Knockdown of Egfl7 inhibits the growth and metastasis of HCCLM3 cells in vivo. (A) The size of liver tumors in the HCCLM3Egfl7RNAi group was dramatically smaller than that of the HCCLM3Egfl7RNAi group. Black arrows: local tumors at the sites of implantation. The volume of liver tumors was calculated as follows: tumor volume (mm3) length width thickness. Relative tumor volume (volume of tumor tissue)/(volume of initially planted tumor tissue). (B) H&E staining was used in the HCCLM3Egfl7RNAi group (a) and the HCCLM3Egfl7RNAi group (b) and immunohistochemistry examination for Egfl7 and phosphorylated FAK expression in tumor was also performed in the HCCLM3Egfl7RNAi group (c,e) and the HCCLM3Egfl7RNAi group (d,f). Original magnification 400. (C) Metastasis of HCCLM3: (a) intrahepatic metastasis of HCCLM3Egfl7RNAi group (yellow arrow, local tumor; green arrows, metastatic nodules); (b) pulmonary metastasis of HCCLM3Egfl7RNAi group (blue arrow, metastatic HCCLM3 cells in the lung tissues of the mice, H&E stain, original magnification 400). (D) MVD was calculated in tumors of the HCCLM3Egfl7RNAi group (a) and HCCLM3Egfl7RNAi group (b) and the results showed that the average MVD in tumors of the HCCLM3Egfl7RNAi group was significantly lower than that in the HCCLM3Egfl7RNAi group (24 11 versus 49 23, P 0.05). *P 0.05.

Article Snippet: The blotted membranes were incubated antihuman Egfl7 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), antihuman FAK antibody (Santa Cruz), or antihuman phosphorylated FAK antibody (Santa Cruz) and then secondary antibody (KPL, Gaithersburg, MD) in order.

Techniques: Knockdown, In Vivo, Staining, Immunohistochemistry, Expressing

Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK (pFAK, Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.

Journal: The FEBS journal

Article Title: Syndecan-1 modulates β-integrin-dependent and interleukin-6-dependent functions in breast cancer cell adhesion, migration, and resistance to irradiation.

doi: 10.1111/febs.12111

Figure Lengend Snippet: Fig. 4. IL-6 modulates Sdc-1-dependent changes in FAK and NF-jB signaling. MDA-MB-231 cells were subjected to control or Sdc-1 siRNA treatment, and incubated with 25 ngmL1 IL-6 for 0, 10 or 30 min; this was followed by western blot analysis of activated FAK (pFAK, Y925) or activated NF-jB (pNF-jB), respectively. Data were normalized to tubulin expression. (A) In control cells, IL-6 treatment transiently increased constitutive FAK activation after 10 min, and this was followed by a decrease after 30 min of incubation. In contrast, Sdc-1 siRNA depletion led to increased basal FAK activation [10]. IL-6 treatment led to transient normalization of FAK phosphorylation to untreated control levels after 10 min of stimulation, and increased levels were restored after 30 min. Top panel: representative western blot. Bottom panel: quantitative analysis. n 3, *P < 0.05, **P < 0.005. (B) Constitutive NF-jB activation in MDA-MB-231 cells was inhibited by IL-6 treatment both in control and in Sdc-1-depleted cells. Sdc-1 siRNA knockdown resulted in a significantly lower degree of NF-jB activation than in controls. n 3, *P < 0.05, **P < 0.01, ***P < 0.001. Sdc-1si, Sdc-1 siRNA.

Article Snippet: The antibodies pNFjB p65 (ser596, 39H1), pFAK (Tyr925) and FAK were from Cell Signaling (Beverly, MA, USA).

Techniques: Control, Incubation, Western Blot, Expressing, Activation Assay, Phospho-proteomics, Knockdown

LIMCH1 depletion affects the formation of focal adhesions in the cell center and phosphorylation of FAK. (A) Binary images of focal adhesions stained with anti-paxillin antibody in siRNA-treated HeLa cells were processed using ImageJ software. Cell outlines (red) and central areas (green) were defined by staining with phalloidin and NM-IIB, respectively. (B) Quantification of the number of total focal adhesions. (C) The ratio of focal adhesions was measured by dividing the number of central or peripheral adhesions by the number of total focal adhesion. In B and C, results were analyzed using ImageJ software ( n = 118–142 cells in three independent experiments, mean ± SD, * p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). (D) Cell extracts from siRNA-treated cells were probed with anti-pFAK Y397 and FAK antibodies. (E) Relative levels of pFAK Y397 shown in D ( n = 4, mean ± SD, normalized to siRNA control, ** p < 0.001, two-tailed t test).

Journal: Molecular Biology of the Cell

Article Title: LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration

doi: 10.1091/mbc.E15-04-0218

Figure Lengend Snippet: LIMCH1 depletion affects the formation of focal adhesions in the cell center and phosphorylation of FAK. (A) Binary images of focal adhesions stained with anti-paxillin antibody in siRNA-treated HeLa cells were processed using ImageJ software. Cell outlines (red) and central areas (green) were defined by staining with phalloidin and NM-IIB, respectively. (B) Quantification of the number of total focal adhesions. (C) The ratio of focal adhesions was measured by dividing the number of central or peripheral adhesions by the number of total focal adhesion. In B and C, results were analyzed using ImageJ software ( n = 118–142 cells in three independent experiments, mean ± SD, * p < 0.001, one-way ANOVA, Tukey’s multiple comparison test). (D) Cell extracts from siRNA-treated cells were probed with anti-pFAK Y397 and FAK antibodies. (E) Relative levels of pFAK Y397 shown in D ( n = 4, mean ± SD, normalized to siRNA control, ** p < 0.001, two-tailed t test).

Article Snippet: The following antibodies were purchased: rabbit anti-MRLC (3672), mouse anti-pMRLC S19 (3675), and rabbit anti-pMRLC S19/T18 (3674) from Cell Signaling (Danvers, MA); mouse anti–actinin-1 (A7811), mouse anti-vinculin (V9264), mouse anti-FLAG (F1804), rabbit anti–NM-IIA (M8064), and rabbit anti-NMIIB (M7939) from Sigma-Aldrich; goat anti–actinin-4 (sc-49333), rabbit anti-paxillin (sc-5574), and rabbit anti-FAK (sc-558) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit anti-pFAK Y397 (700255) from Invitrogen (Carlsbad, CA); rabbit anti-NM18A was a kind gift from Jau-Song Yu (Chang Gung University, Taipei, Taiwan).

Techniques: Staining, Software, Two Tailed Test