rabbit polyclonal anti klf6  (Santa Cruz Biotechnology)

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 protein and mRNA expression in melanoma cell lines. A) Level of KLF6 protein in normal melanocytes (NHEM) and melanoma cell lines established from primary tumors at the radial (WM35 and WM3211), vertical (WM115, WM98.1, and WM278), and metastatic (UACC 903 and SK-MEL-24) growth phase of melanoma progression. Data are levels of KLF6 protein normalized to that of extracellular-related kinase 2 (the control for protein loading), which were averaged from three independent experiments. # = P = .014; & = P = .22, * = P < .001 (F7,16 = 14.15, two-sided one-way analysis of variance [ANOVA] test followed by Dunnett multiple comparison test to compare melanocyte control cells with WM35, WM3211, WM98, WM115, WM278, UACC 903, and SK-MEL-24 cells); arrows = decreased expression of KLF6 compared with normal melanocytes. B) KLF6 mRNA levels in control UACC 903 and 10ER4S.2/1 cells and in 10E6/3, 10E6/11, and 10E6/18 hybrid cells with the tumor suppressor gene fragment, as well as WM35 and SK-MEL-24 melanoma cells. Human GAPDH mRNA was the internal control, and level of KLF6 mRNA was normalized to that of GAPDH mRNA. Data are the mean of triplicate samples from three independent experiments. Square bracket = percentage of increased expression in hybrid cells; * = P < .001 (F4,40 = 27.33, two-sided one-way ANOVA test followed by Dunnett multiple comparison test to compare UACC 903 control with 10E6/3, 10E6/11, and 10E6/18 hybrid cells); error bars = 95% confidence intervals. Experiments were repeated three times.

Article Snippet: Briefly, we used the following primary antibodies: mouse monoclonal anti-hemagglutinin A (1:2000 dilution), rabbit polyclonal anti-human KLF6 (1:500 dilution), rabbit polyclonal anti-human cyclin D1 (1:1000 dilution), mouse monoclonal anti-human extracellular-related kinase 2 (ERK2) (1:2000 dilution), and goat polyclonal anti-human α-enolase (1:1000 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit polyclonal anti-human phosphorylated extracellular-related kinase (1:1000 dilution) and mouse monoclonal anti-human CDK4 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: Ectopic expression of KLF6 and viability of UACC 903 and SK-MEL-24 melanoma cells on type I collagen and on plastic. Expression of wild-type and mutant KLF6 protein in melanoma cells on type I collagen. A, Upper) Immunoblots of total lysates collected from UACC 903 or SK-MEL-24 cells ectopically expressing wild-type KLF6, hemagluttinin A (HA)-tagged KLF6 (HA3KLF6), and mutant forms of KLF6. Cells carrying empty puro vectors were the control. Blots were probed with antibodies against the HA tag or wild-type KLF6 protein. Intact = ectopical expression of wild-type HA-KLF6; truncated = mutant KLF6 form HA-W162X. Endogenous levels of KLF6 were probed with an antibody against KLF6 protein. α-Enolase was the control for equal protein loading. A, Lower) Relative levels of KLF6 protein. Levels of KLF6 protein in blots in panel (A) were normalized to that of α-enolase, and the relative expression was plotted. Endogenous levels of KLF6 in UACC 903 vector control were set as 1 (dash line). The normal physiological range of KLF6 protein is 1.5 as shown in WM35 cells, which has similar expression levels as normal melanocytes. Blots were repeated twice, and both showed similar results. B) Expression of KLF6 and viability of melanoma cells on collagen I. Viability of UACC 903 or SK-MEL-24 cells ectopically expressing wild-type or HA-tagged KLF6 (HA3KLF6) was assessed in type I collagen cell culture and compared with that of control cells expressing inactive (HA3K209R and HA3W162X) protein or carrying the empty puro vector, by use of 3-(4,5-dimethylthiazon-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Data are the mean value from eight samples; the experiment was repeated three times. * = P < .001 (F4,35 = 641.17 for UACC 903 cells and F4,35 = 102.76 for SK-MEL-24 cells; two-sided one-way analysis of variance test followed by Dunnett multiple comparison test to compare UACC 903 or SK-MEL-24 puro vector control cells with KLF6 ectopically expressing cells); error bars = 95% confidence intervals.

Article Snippet: Briefly, we used the following primary antibodies: mouse monoclonal anti-hemagglutinin A (1:2000 dilution), rabbit polyclonal anti-human KLF6 (1:500 dilution), rabbit polyclonal anti-human cyclin D1 (1:1000 dilution), mouse monoclonal anti-human extracellular-related kinase 2 (ERK2) (1:2000 dilution), and goat polyclonal anti-human α-enolase (1:1000 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit polyclonal anti-human phosphorylated extracellular-related kinase (1:1000 dilution) and mouse monoclonal anti-human CDK4 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Cell Culture, MTS Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 small interfering RNA (siRNA), melanoma cells containing chromosome 10p15 fragments, and viability on type I collagen of UACC 903, 10E6/3, 10E6/11, 10E6/18, and 10ER4S.2/1 cells. A) KLF6 siRNA and KLF6 protein expression. siRNA (100 pmol) of was introduced by nucleofection with an Amaxa Nucleofector, and 2 days later, protein lysates were prepared and KLF6 protein expression was assessed by immunoblot analysis with KLF6 antibody. Untransfected or cells nucleofected with scrambled siRNA were used as controls, and α-enolase was the control for equal protein loading. Arrow = reduction of KLF6 expression compared with control. Experiments were repeated three times. B) Cell viability at day 5 of cells cultured on type I collagen. Data are the mean value of eight samples; the experiment was repeated three times. * = P < .001 (F2,21 = 361.7 for 10E6/3 cells, 161.2 for 10E6/11 cells, and 260.6 for 10E6/18 cells; two-sided one-way analysis of variance test followed by Dunnett multiple comparison test to compare the effect of siRNA-mediated targeting KLF6 with that of untransfected and scrambled siRNA controls); error bars = 95% confidence intervals; arrow = increased cell viability compared with controls.

Article Snippet: Briefly, we used the following primary antibodies: mouse monoclonal anti-hemagglutinin A (1:2000 dilution), rabbit polyclonal anti-human KLF6 (1:500 dilution), rabbit polyclonal anti-human cyclin D1 (1:1000 dilution), mouse monoclonal anti-human extracellular-related kinase 2 (ERK2) (1:2000 dilution), and goat polyclonal anti-human α-enolase (1:1000 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit polyclonal anti-human phosphorylated extracellular-related kinase (1:1000 dilution) and mouse monoclonal anti-human CDK4 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA).

Techniques: Small Interfering RNA, Expressing, Western Blot, Cell Culture

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: Mitogen-activated protein (MAP) kinase pathway signaling, KLF6 expression, and culture on type I collagen or plastic in UACC 903 or SK-MEL-24. A) Cell lines derived from UACC 903 melanoma cells. B) Cell lines derived from SK-MEL-24 melanoma cells. Levels of phosphorylated extracelluar-related kinase (pErk1/2) and cyclin D1 were assessed in lysates collected from UACC 903 and SK-MEL-24 cells ectopically expressing wild-type KLF6, HA-KLF6, or mutant forms of KLF6 by immunoblot analysis with anti-phosphorylated Erk1/2 and anti-cyclin D1 antibodies. Numerical values below each blot are the normalized averages of pErk1/2 and cyclin D1 expression from two independent experiments. Averages were normalized to the value for α-enolase (control for protein loading). Experiments were repeated two times.

Article Snippet: Briefly, we used the following primary antibodies: mouse monoclonal anti-hemagglutinin A (1:2000 dilution), rabbit polyclonal anti-human KLF6 (1:500 dilution), rabbit polyclonal anti-human cyclin D1 (1:1000 dilution), mouse monoclonal anti-human extracellular-related kinase 2 (ERK2) (1:2000 dilution), and goat polyclonal anti-human α-enolase (1:1000 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit polyclonal anti-human phosphorylated extracellular-related kinase (1:1000 dilution) and mouse monoclonal anti-human CDK4 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Derivative Assay, Mutagenesis, Western Blot

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 protein and mRNA expression in human melanomas and human nevi. A) Immunohistochemical analysis of KLF6 protein expression in melanoma tumors and nevi. Formalin-fixed paraffin-embedded sections of atypical nevi, primary melanomas of greater than 0.75 mm in depth, and metastatic melanomas were stained with hematoxylin–eosin (H&E) or for KLF6 protein expression by immunohistochemistry with anti-KLF6 and secondary avidin–biotin horseradish peroxidase anti-rabbit IgG antibody. Brown staining = KLF6 protein; arrowheads = KLF6 staining. Staining of KLF6 in epidermis served as the positive control. Bottom row is a higher magnification of the section in middle row. Scale bars = 50 μm. B) KLF6 protein expression in xenografted UACC 903 tumors at day 17. Arrowheads = KLF6 staining; arrows = exterior and interior of tumors at high magnification in the right panel. Scale bars = 50 μm. C) KLF6 protein expression in human metastatic melanomas. Protein lysates were prepared from of 29 human melanoma tumors (numbered 1–29) and analyzed for expression of KLF6, ITIH5 (a gene also located at 10p15), PTEN (a gene located at 10q23), and extracellular-related kinase2 (Erk2) proteins. PTEN protein status: + = present moderate expression; ++ = high expression; − = absent. Erk2 was the control for equal protein loading. WM35 and SK-MEL-24 cell lysates served as positive and negative controls for high and low KLF6 protein expression, respectively. Experiments were repeated three times. D) KLF6 mRNA expression in five human melanoma tumors that lacked KLF6 protein expression. KLF6 mRNA levels were measured with a quantitative real-time polymerase chain reaction in triplicate samples and in three experiments and were compared with that in WM35 cells, which have KLF6 protein levels similar to that in normal melanocytes. Human GAPDH sequences were amplified for use as an internal control and for normalization of KLF6 mRNA. The mRNA from WM35 cells served as a positive control for high KLF6 mRNA expression. Data are the mean value from triplicate sample, and each experiment was repeated three times. Error bars = 95% confidence intervals.

Article Snippet: Briefly, we used the following primary antibodies: mouse monoclonal anti-hemagglutinin A (1:2000 dilution), rabbit polyclonal anti-human KLF6 (1:500 dilution), rabbit polyclonal anti-human cyclin D1 (1:1000 dilution), mouse monoclonal anti-human extracellular-related kinase 2 (ERK2) (1:2000 dilution), and goat polyclonal anti-human α-enolase (1:1000 dilution) from Santa Cruz Biotechnology (Santa Cruz, CA); and rabbit polyclonal anti-human phosphorylated extracellular-related kinase (1:1000 dilution) and mouse monoclonal anti-human CDK4 (1:1000 dilution) from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Avidin-Biotin Assay, Positive Control, Real-time Polymerase Chain Reaction, Amplification

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 protein and mRNA expression in melanoma cell lines. A) Level of KLF6 protein in normal melanocytes (NHEM) and melanoma cell lines established from primary tumors at the radial (WM35 and WM3211), vertical (WM115, WM98.1, and WM278), and metastatic (UACC 903 and SK-MEL-24) growth phase of melanoma progression. Data are levels of KLF6 protein normalized to that of extracellular-related kinase 2 (the control for protein loading), which were averaged from three independent experiments. # = P = .014; & = P = .22, * = P < .001 (F7,16 = 14.15, two-sided one-way analysis of variance [ANOVA] test followed by Dunnett multiple comparison test to compare melanocyte control cells with WM35, WM3211, WM98, WM115, WM278, UACC 903, and SK-MEL-24 cells); arrows = decreased expression of KLF6 compared with normal melanocytes. B) KLF6 mRNA levels in control UACC 903 and 10ER4S.2/1 cells and in 10E6/3, 10E6/11, and 10E6/18 hybrid cells with the tumor suppressor gene fragment, as well as WM35 and SK-MEL-24 melanoma cells. Human GAPDH mRNA was the internal control, and level of KLF6 mRNA was normalized to that of GAPDH mRNA. Data are the mean of triplicate samples from three independent experiments. Square bracket = percentage of increased expression in hybrid cells; * = P < .001 (F4,40 = 27.33, two-sided one-way ANOVA test followed by Dunnett multiple comparison test to compare UACC 903 control with 10E6/3, 10E6/11, and 10E6/18 hybrid cells); error bars = 95% confidence intervals. Experiments were repeated three times.

Article Snippet: Sections (5 μm thick) from 17 samples of atypical nevi, sections (<0.75 mm thick) from five samples of melanomas, and sections (5 μm thick) from four samples of malignant melanomas were stained with rabbit polyclonal anti-human KLF6 antibody (Santa Cruz Biotechnology) that had been diluted 1:50 in 1.5% goat serum and incubated at 4°C overnight.

Techniques: Expressing, Control, Comparison

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: Ectopic expression of KLF6 and viability of UACC 903 and SK-MEL-24 melanoma cells on type I collagen and on plastic. Expression of wild-type and mutant KLF6 protein in melanoma cells on type I collagen. A, Upper) Immunoblots of total lysates collected from UACC 903 or SK-MEL-24 cells ectopically expressing wild-type KLF6, hemagluttinin A (HA)-tagged KLF6 (HA3KLF6), and mutant forms of KLF6. Cells carrying empty puro vectors were the control. Blots were probed with antibodies against the HA tag or wild-type KLF6 protein. Intact = ectopical expression of wild-type HA-KLF6; truncated = mutant KLF6 form HA-W162X. Endogenous levels of KLF6 were probed with an antibody against KLF6 protein. α-Enolase was the control for equal protein loading. A, Lower) Relative levels of KLF6 protein. Levels of KLF6 protein in blots in panel (A) were normalized to that of α-enolase, and the relative expression was plotted. Endogenous levels of KLF6 in UACC 903 vector control were set as 1 (dash line). The normal physiological range of KLF6 protein is 1.5 as shown in WM35 cells, which has similar expression levels as normal melanocytes. Blots were repeated twice, and both showed similar results. B) Expression of KLF6 and viability of melanoma cells on collagen I. Viability of UACC 903 or SK-MEL-24 cells ectopically expressing wild-type or HA-tagged KLF6 (HA3KLF6) was assessed in type I collagen cell culture and compared with that of control cells expressing inactive (HA3K209R and HA3W162X) protein or carrying the empty puro vector, by use of 3-(4,5-dimethylthiazon-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Data are the mean value from eight samples; the experiment was repeated three times. * = P < .001 (F4,35 = 641.17 for UACC 903 cells and F4,35 = 102.76 for SK-MEL-24 cells; two-sided one-way analysis of variance test followed by Dunnett multiple comparison test to compare UACC 903 or SK-MEL-24 puro vector control cells with KLF6 ectopically expressing cells); error bars = 95% confidence intervals.

Article Snippet: Sections (5 μm thick) from 17 samples of atypical nevi, sections (<0.75 mm thick) from five samples of melanomas, and sections (5 μm thick) from four samples of malignant melanomas were stained with rabbit polyclonal anti-human KLF6 antibody (Santa Cruz Biotechnology) that had been diluted 1:50 in 1.5% goat serum and incubated at 4°C overnight.

Techniques: Expressing, Mutagenesis, Western Blot, Control, Plasmid Preparation, Cell Culture, MTS Assay, Comparison

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 small interfering RNA (siRNA), melanoma cells containing chromosome 10p15 fragments, and viability on type I collagen of UACC 903, 10E6/3, 10E6/11, 10E6/18, and 10ER4S.2/1 cells. A) KLF6 siRNA and KLF6 protein expression. siRNA (100 pmol) of was introduced by nucleofection with an Amaxa Nucleofector, and 2 days later, protein lysates were prepared and KLF6 protein expression was assessed by immunoblot analysis with KLF6 antibody. Untransfected or cells nucleofected with scrambled siRNA were used as controls, and α-enolase was the control for equal protein loading. Arrow = reduction of KLF6 expression compared with control. Experiments were repeated three times. B) Cell viability at day 5 of cells cultured on type I collagen. Data are the mean value of eight samples; the experiment was repeated three times. * = P < .001 (F2,21 = 361.7 for 10E6/3 cells, 161.2 for 10E6/11 cells, and 260.6 for 10E6/18 cells; two-sided one-way analysis of variance test followed by Dunnett multiple comparison test to compare the effect of siRNA-mediated targeting KLF6 with that of untransfected and scrambled siRNA controls); error bars = 95% confidence intervals; arrow = increased cell viability compared with controls.

Article Snippet: Sections (5 μm thick) from 17 samples of atypical nevi, sections (<0.75 mm thick) from five samples of melanomas, and sections (5 μm thick) from four samples of malignant melanomas were stained with rabbit polyclonal anti-human KLF6 antibody (Santa Cruz Biotechnology) that had been diluted 1:50 in 1.5% goat serum and incubated at 4°C overnight.

Techniques: Small Interfering RNA, Expressing, Western Blot, Control, Cell Culture, Comparison

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: Mitogen-activated protein (MAP) kinase pathway signaling, KLF6 expression, and culture on type I collagen or plastic in UACC 903 or SK-MEL-24. A) Cell lines derived from UACC 903 melanoma cells. B) Cell lines derived from SK-MEL-24 melanoma cells. Levels of phosphorylated extracelluar-related kinase (pErk1/2) and cyclin D1 were assessed in lysates collected from UACC 903 and SK-MEL-24 cells ectopically expressing wild-type KLF6, HA-KLF6, or mutant forms of KLF6 by immunoblot analysis with anti-phosphorylated Erk1/2 and anti-cyclin D1 antibodies. Numerical values below each blot are the normalized averages of pErk1/2 and cyclin D1 expression from two independent experiments. Averages were normalized to the value for α-enolase (control for protein loading). Experiments were repeated two times.

Article Snippet: Sections (5 μm thick) from 17 samples of atypical nevi, sections (<0.75 mm thick) from five samples of melanomas, and sections (5 μm thick) from four samples of malignant melanomas were stained with rabbit polyclonal anti-human KLF6 antibody (Santa Cruz Biotechnology) that had been diluted 1:50 in 1.5% goat serum and incubated at 4°C overnight.

Techniques: Expressing, Derivative Assay, Mutagenesis, Western Blot, Control

Journal: JNCI Journal of the National Cancer Institute

Article Title: KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

doi: 10.1093/jnci/djq218

Figure Lengend Snippet: KLF6 protein and mRNA expression in human melanomas and human nevi. A) Immunohistochemical analysis of KLF6 protein expression in melanoma tumors and nevi. Formalin-fixed paraffin-embedded sections of atypical nevi, primary melanomas of greater than 0.75 mm in depth, and metastatic melanomas were stained with hematoxylin–eosin (H&E) or for KLF6 protein expression by immunohistochemistry with anti-KLF6 and secondary avidin–biotin horseradish peroxidase anti-rabbit IgG antibody. Brown staining = KLF6 protein; arrowheads = KLF6 staining. Staining of KLF6 in epidermis served as the positive control. Bottom row is a higher magnification of the section in middle row. Scale bars = 50 μm. B) KLF6 protein expression in xenografted UACC 903 tumors at day 17. Arrowheads = KLF6 staining; arrows = exterior and interior of tumors at high magnification in the right panel. Scale bars = 50 μm. C) KLF6 protein expression in human metastatic melanomas. Protein lysates were prepared from of 29 human melanoma tumors (numbered 1–29) and analyzed for expression of KLF6, ITIH5 (a gene also located at 10p15), PTEN (a gene located at 10q23), and extracellular-related kinase2 (Erk2) proteins. PTEN protein status: + = present moderate expression; ++ = high expression; − = absent. Erk2 was the control for equal protein loading. WM35 and SK-MEL-24 cell lysates served as positive and negative controls for high and low KLF6 protein expression, respectively. Experiments were repeated three times. D) KLF6 mRNA expression in five human melanoma tumors that lacked KLF6 protein expression. KLF6 mRNA levels were measured with a quantitative real-time polymerase chain reaction in triplicate samples and in three experiments and were compared with that in WM35 cells, which have KLF6 protein levels similar to that in normal melanocytes. Human GAPDH sequences were amplified for use as an internal control and for normalization of KLF6 mRNA. The mRNA from WM35 cells served as a positive control for high KLF6 mRNA expression. Data are the mean value from triplicate sample, and each experiment was repeated three times. Error bars = 95% confidence intervals.

Article Snippet: Sections (5 μm thick) from 17 samples of atypical nevi, sections (<0.75 mm thick) from five samples of melanomas, and sections (5 μm thick) from four samples of malignant melanomas were stained with rabbit polyclonal anti-human KLF6 antibody (Santa Cruz Biotechnology) that had been diluted 1:50 in 1.5% goat serum and incubated at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Avidin-Biotin Assay, Positive Control, Control, Real-time Polymerase Chain Reaction, Amplification