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Figure 2 | Staphylococcus aureus activates nuclear factor-kappa B (NF-κB) and cAMP response element-binding <t>(CREB)</t> phosphorylation and induces NF-κB nuclear translocation. Bovine endothelial cells were left uninfected (−) or infected at a multiplicity of infection of 20 CFU/cell of S. aureus for 20–120 min. After infection, the total protein (nuclear + cytoplasmic) of (A,B) or nuclear- and cytoplasmic-enriched fractions (C) was analyzed by Western blotting to detect the relative abundance of phosphorylated NF-κB p65 at Ser536 (A), phosphorylated CREB at Ser133 (B), or NF-κB p65 (C). The detection of total unphosphorylated NF-κB p65 (A), total unphosphorylated CREB (B), and β-actin (A,B) was performed to ensure equal protein loading. GAPDH and Laminin A/C were detected as protein markers to ensure equal protein loading for cytoplasmic- and nuclear-enriched fractions, respectively (C). Error bars in graphs A and B represent the mean ± SEM (n = 3) of densitometric values. *P < 0.05, **P < 0.01 referred to the uninfected control value.
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Figure 2 | Staphylococcus aureus activates nuclear factor-kappa B (NF-κB) and cAMP response element-binding (CREB) phosphorylation and induces NF-κB nuclear translocation. Bovine endothelial cells were left uninfected (−) or infected at a multiplicity of infection of 20 CFU/cell of S. aureus for 20–120 min. After infection, the total protein (nuclear + cytoplasmic) of (A,B) or nuclear- and cytoplasmic-enriched fractions (C) was analyzed by Western blotting to detect the relative abundance of phosphorylated NF-κB p65 at Ser536 (A), phosphorylated CREB at Ser133 (B), or NF-κB p65 (C). The detection of total unphosphorylated NF-κB p65 (A), total unphosphorylated CREB (B), and β-actin (A,B) was performed to ensure equal protein loading. GAPDH and Laminin A/C were detected as protein markers to ensure equal protein loading for cytoplasmic- and nuclear-enriched fractions, respectively (C). Error bars in graphs A and B represent the mean ± SEM (n = 3) of densitometric values. *P < 0.05, **P < 0.01 referred to the uninfected control value.

Journal: Frontiers in immunology

Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus .

doi: 10.3389/fimmu.2018.00092

Figure Lengend Snippet: Figure 2 | Staphylococcus aureus activates nuclear factor-kappa B (NF-κB) and cAMP response element-binding (CREB) phosphorylation and induces NF-κB nuclear translocation. Bovine endothelial cells were left uninfected (−) or infected at a multiplicity of infection of 20 CFU/cell of S. aureus for 20–120 min. After infection, the total protein (nuclear + cytoplasmic) of (A,B) or nuclear- and cytoplasmic-enriched fractions (C) was analyzed by Western blotting to detect the relative abundance of phosphorylated NF-κB p65 at Ser536 (A), phosphorylated CREB at Ser133 (B), or NF-κB p65 (C). The detection of total unphosphorylated NF-κB p65 (A), total unphosphorylated CREB (B), and β-actin (A,B) was performed to ensure equal protein loading. GAPDH and Laminin A/C were detected as protein markers to ensure equal protein loading for cytoplasmic- and nuclear-enriched fractions, respectively (C). Error bars in graphs A and B represent the mean ± SEM (n = 3) of densitometric values. *P < 0.05, **P < 0.01 referred to the uninfected control value.

Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift 3 Silva-García et al. GSK3α Regulates NF-κB and CREB Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 92 from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay, Infection, Western Blot, Control

Figure 4 | The constitutive activity of glycogen synthase kinase 3 (GSK3α/β) isoforms regulates nuclear factor-kappa B (NF-κB) and cAMP response element- binding (CREB) activity. Bovine endothelial cells (BECs) were left untreated and uninfected (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) (A) or the Akt inhibitor Akt-IV (1 µM) (B) for 1 h before infection at a multiplicity of infection of 20 CFU/cell of Staphylococcus aureus for the indicated times. Controls pretreated with the inhibitors alone but not infected were also included. After infection, the relative abundance of phosphorylated forms of NF-κB p65 at Ser536 and CREB at Ser133 was detected by Western blotting. Unphosphorylated NF-κB p65 and CREB, and GAPDH were detected to ensure equal protein loading. (C) BECs were left untreated and uninfected (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) for 1 h before infection with 20 CFU/cell of S. aureus for the indicated time. After infection, nuclear- and cytoplasmic-enriched fractions were obtained, and unphosphorylated NF-κB p65 was detected. GAPDH and Laminin A/C were detected as protein markers to ensure equal protein loading for cytoplasmic- and nuclear-enriched fractions, respectively. Blots are representative of three independent experiments.

Journal: Frontiers in immunology

Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus .

doi: 10.3389/fimmu.2018.00092

Figure Lengend Snippet: Figure 4 | The constitutive activity of glycogen synthase kinase 3 (GSK3α/β) isoforms regulates nuclear factor-kappa B (NF-κB) and cAMP response element- binding (CREB) activity. Bovine endothelial cells (BECs) were left untreated and uninfected (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) (A) or the Akt inhibitor Akt-IV (1 µM) (B) for 1 h before infection at a multiplicity of infection of 20 CFU/cell of Staphylococcus aureus for the indicated times. Controls pretreated with the inhibitors alone but not infected were also included. After infection, the relative abundance of phosphorylated forms of NF-κB p65 at Ser536 and CREB at Ser133 was detected by Western blotting. Unphosphorylated NF-κB p65 and CREB, and GAPDH were detected to ensure equal protein loading. (C) BECs were left untreated and uninfected (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) for 1 h before infection with 20 CFU/cell of S. aureus for the indicated time. After infection, nuclear- and cytoplasmic-enriched fractions were obtained, and unphosphorylated NF-κB p65 was detected. GAPDH and Laminin A/C were detected as protein markers to ensure equal protein loading for cytoplasmic- and nuclear-enriched fractions, respectively. Blots are representative of three independent experiments.

Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift 3 Silva-García et al. GSK3α Regulates NF-κB and CREB Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 92 from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

Techniques: Activity Assay, Binding Assay, Infection, Western Blot

Figure 6 | The glycogen synthase kinase 3 (GSK3α/β) isoforms regulate the interaction of nuclear factor-kappa B (NF-κB) and cAMP response element binding (CREB) with the transcriptional co-activator CREB-binding protein (CBP). (A,B) Cells were left uninfected and untreated (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) or the Akt inhibitor Akt-IV (1 µM) for 1 h and infected with 20 CFU/cell of Staphylococcus aureus for 40 (A) or 120 min (B). The total protein was extracted (input fraction) for immunoprecipitation (IP) of CBP. Eluates obtained were then used to detect (IB) NF-κB or CREB by Western blotting. A negative control with an iso-type IgG control was also included. NF-κB and CREB were detected in the input fractions to ensure equal protein loading. (C,D) Cells were transfected with plasmids containing siRNA sequences against GSK3α, GSK3β, or with control vector as indicated for 5 days. Then, siRNA of GSK3 isoforms was evaluated by Western blotting. (D) GSK3α/β-silenced cells were infected with S. aureus for 120 min, and then CBP protein was immunoprecipitated from cell lysates; NF-κB and CREB proteins were detected in the CBP immunoprecipitates by Western blotting. IgG control was included and the total CREB and NF-κB in input cell lysates are also shown.

Journal: Frontiers in immunology

Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus .

doi: 10.3389/fimmu.2018.00092

Figure Lengend Snippet: Figure 6 | The glycogen synthase kinase 3 (GSK3α/β) isoforms regulate the interaction of nuclear factor-kappa B (NF-κB) and cAMP response element binding (CREB) with the transcriptional co-activator CREB-binding protein (CBP). (A,B) Cells were left uninfected and untreated (−) or pretreated with the GSK3α/β inhibitor SB216763 (10 µM) or the Akt inhibitor Akt-IV (1 µM) for 1 h and infected with 20 CFU/cell of Staphylococcus aureus for 40 (A) or 120 min (B). The total protein was extracted (input fraction) for immunoprecipitation (IP) of CBP. Eluates obtained were then used to detect (IB) NF-κB or CREB by Western blotting. A negative control with an iso-type IgG control was also included. NF-κB and CREB were detected in the input fractions to ensure equal protein loading. (C,D) Cells were transfected with plasmids containing siRNA sequences against GSK3α, GSK3β, or with control vector as indicated for 5 days. Then, siRNA of GSK3 isoforms was evaluated by Western blotting. (D) GSK3α/β-silenced cells were infected with S. aureus for 120 min, and then CBP protein was immunoprecipitated from cell lysates; NF-κB and CREB proteins were detected in the CBP immunoprecipitates by Western blotting. IgG control was included and the total CREB and NF-κB in input cell lysates are also shown.

Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift 3 Silva-García et al. GSK3α Regulates NF-κB and CREB Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 92 from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

Techniques: Binding Assay, Infection, Immunoprecipitation, Western Blot, Negative Control, Control, Transfection, Plasmid Preparation

Figure 5 | The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with PLKO.1 vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM (n = 3) of three independent experiments. *P < 0.05, referred to the untransfected control. (B) BECs were infected with 20 CFU/cell of Staphylococcus aureus for 40 min. After infection, the relative abundance of phosphorylated CREB at Ser133 was analyzed by Western blotting. Unphosphorylated CREB and GAPDH were detected to ensure equal protein loading. Error bars in graph represent the mean ± SEM (n = 3) of three independent experiments. *P < 0.05, **P < 0.001 compared to control.

Journal: Frontiers in immunology

Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus .

doi: 10.3389/fimmu.2018.00092

Figure Lengend Snippet: Figure 5 | The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with PLKO.1 vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM (n = 3) of three independent experiments. *P < 0.05, referred to the untransfected control. (B) BECs were infected with 20 CFU/cell of Staphylococcus aureus for 40 min. After infection, the relative abundance of phosphorylated CREB at Ser133 was analyzed by Western blotting. Unphosphorylated CREB and GAPDH were detected to ensure equal protein loading. Error bars in graph represent the mean ± SEM (n = 3) of three independent experiments. *P < 0.05, **P < 0.001 compared to control.

Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift 3 Silva-García et al. GSK3α Regulates NF-κB and CREB Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 92 from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

Techniques: Activity Assay, Binding Assay, Phospho-proteomics, Transfection, Plasmid Preparation, Expressing, Control, Infection, Western Blot

Figure 7 | Staphylococcus aureus-induced expression of interleukin-8 (IL-8) and interleukin-10 (IL-10) is regulated by nuclear factor-kappa B (NF-κB) and cAMP response element binding (CREB). (A,B) Bovine endothelial cells (BECs) were left uninfected and untreated (−) or pretreated with the NF-κB inhibitor Parthenolide (20 µM) for 1 h and then infected with 20 CFU/cell of S. aureus for 6 h. A control group pretreated with 20 µM of Parthenolide was also included. (C,D) BECs were treated with a medium or transfected with a plasmid containing the dominant-negative mutant of CREB-S133A (DN-CREB) or the control plasmid pCF (control vector) for 48 h and then infected with 20 CFU/cell of S. aureus for 6 h. Control-uninfected cells transfected with the DN-CREB or plasmid pCF were included. After infection, the conditioned media were collected in ice-cold tubes and kept frozen at −80°C. Quantitation of IL-8 and IL-10 was done by ELISA, according to the manufacturer’s instructions. Data represent the mean ± SEM (n = 3) from two independent experiments. *P < 0.05, **P < 0.01 as compared to control.

Journal: Frontiers in immunology

Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus .

doi: 10.3389/fimmu.2018.00092

Figure Lengend Snippet: Figure 7 | Staphylococcus aureus-induced expression of interleukin-8 (IL-8) and interleukin-10 (IL-10) is regulated by nuclear factor-kappa B (NF-κB) and cAMP response element binding (CREB). (A,B) Bovine endothelial cells (BECs) were left uninfected and untreated (−) or pretreated with the NF-κB inhibitor Parthenolide (20 µM) for 1 h and then infected with 20 CFU/cell of S. aureus for 6 h. A control group pretreated with 20 µM of Parthenolide was also included. (C,D) BECs were treated with a medium or transfected with a plasmid containing the dominant-negative mutant of CREB-S133A (DN-CREB) or the control plasmid pCF (control vector) for 48 h and then infected with 20 CFU/cell of S. aureus for 6 h. Control-uninfected cells transfected with the DN-CREB or plasmid pCF were included. After infection, the conditioned media were collected in ice-cold tubes and kept frozen at −80°C. Quantitation of IL-8 and IL-10 was done by ELISA, according to the manufacturer’s instructions. Data represent the mean ± SEM (n = 3) from two independent experiments. *P < 0.05, **P < 0.01 as compared to control.

Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift 3 Silva-García et al. GSK3α Regulates NF-κB and CREB Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 92 from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

Techniques: Expressing, Binding Assay, Infection, Control, Transfection, Plasmid Preparation, Dominant Negative Mutation, Quantitation Assay, Enzyme-linked Immunosorbent Assay