Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: β-actin was monitored as the internal standard to ensure similar gel loading of the starting materials in each sample. Blot images were cropped for comparison (E). Electroacupuncture treatment significantly increased the levels of phospho-Akt protein, HO-1 protein, Nrf2 total and nucleoprotein compared with group L or group SEL. In wortmannin-treated rabbits, phosphorylation of Akt was significantly decreased while HO-1 protein, Nrf2 total and nucleoprotein was slightly decreased compared with group EL. All values were expressed as mean ±SD (n = 10; *P<0 . 05; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons). The blots were representative of three independent experiments.

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Comparison, Phospho-proteomics

Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: (original magnification×400): (A) The pictures of immunofluorescence staining. Green stands for Nrf2-FITC stained sections, while blue stands for images of DAPI stained nuclei. The overlay color of blue staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. (B) Quantification of nuclear localization of Nrf2 protein among six groups. Electroacupuncture protocols dramatically increased translocation of Nrf2 from cytoplasm into nucleus compared with group L or SEL. To some degree, pretreatment with wortmannin counteracted nuclear accumulation of Nrf2 protein, while wortmannin alone had no effects. Data were representative of three independent experiments. Values were expressed as mean ± SD (n = 10; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons).

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Immunofluorescence, Staining, Translocation Assay

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 1. RSK1, via its N-terminal kinase domain, interacts with PKARI. A, shown is a schematic of rat RSK1 and HA-tagged RSK1 constructs used; numbers represent the amino acids in rat RSK1. B–E, shown is pulldown of HA-tagged RSK1 or its polypeptides with cAMP-agarose. HEK293T cells were transfected to express HA-tagged RSK1 or its polypeptides. After depriving of serum overnight, cells were lysed and incu- bated with cAMP-agarose to pull down PKARI in the presence and absence of 50 mM cAMP as indicated. The proteins in the complex were monitored using anti-HA, anti-PKARI, or anti-RSK1 antibodies. IB, immunoblot. B, PKARI binds to RSK1 fragments containing the N-terminal part of RSK1. C, HA-RSK1-(1–317) competes with endogenousRSK1forbindingtoPKARI.ThepanelontherightshowsquantificationofbandintensitiesofRSK1 as a ratio of band intensities of PKARI (mean S.E.) from three experiments. *, p 0.05 as compared with the control.D,theNTKofRSK1bindstoPKARI.E,substitutionofSer-221ofRSK1withanegativelychargedresidue abrogates the interaction between RSK1 and PKARI. Representatives of three similar experiments are shown for all panels. WCL, whole cell lysates.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Construct, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 2. RSK1 binds to the pseudosubstrate region of PKARI. A, shown is a schematic of bovine PKARI; numbers represent the amino acids in bovine PKARI. B, the truncated PKARI (deletion of amino acids 1–91, PKARI91) retains the ability to interact with RSK1. Pure PKARI or PKARI91 (10 pmol each) was incubated with glutathione-Sepharose prebound with GST or GST-RSK1-(1–317) (5 g). The amounts of PKARI or PKARI91 in the pulldown complex were detected with anti-PKARI antibody. GST or GST-RSK1-(1–317) was stained with Coomassie Blue. IB, immunoblot. C, substitution of Arg-93/94 or Arg-95/96 on PKARI to Ala selectively abrogates the interactions of PKARI with RSK1 and PKAc. HEK293T cells were transfected with plasmids expressing C-terminal fusion of Wt-PKARI, PKARI (R93A/R94A), or PKARI (R95A/R96A) with enhanced yellow fluorescent protein (wt-PKARI-EYFP, PKARI (R93A/R94A)-EYFP, and PKARI (R95A/R96A)- EYFP, respectively). Cell lysates were immunoprecipitated (IP) with anti-RSK1 or PKAc antibody. The immune complex was probed with anti-EYFP, PKARI, RSK1, or PKAc antibodies. WCL, whole cell lysate. D, the peptide corresponding to the PKARI pseudosubstrate region (Wt-PS) competes for the interaction of PKARI with RSK1. GST-RSK1-(1–317) (5 g) prebound to glutathione-Sepharose was incubated with PKARI peptides, Wt-PS, or Mut-PS at the concentrations indicated at 4 °C for 15 min before being mixed with PKARI (10 pmol). GST-RSK1-(1–317) was stained with Coomassie Blue. The panel on the right shows the quantification of relative intensities of PKARI bands as a ratio of GST-RSK1 bands from two identical experiments. Wt-PS, peptide with wild-type PKARI pseudosubstrate sequence KGRRRRGAI). Mut-PS, peptide with mutated PKARI pseudosub- strate sequence (KGAARRGAI).

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Incubation, Staining, Western Blot, Transfection, Expressing, Immunoprecipitation, Sequencing

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE3.AffinitiesofPKAcandRSK1forPKARI.PKARIwasimmobilized on GLC sensor chips of the Bio-Rad Proteon XPR36 instrument, and binding isotherms were monitored by infusing the different concentrations of PKAc (A), GST-full-length RSK1 (GST-FL-RSK1) (B), or GST-RSK1-(1–317) (C) for the 600-s periods (denoted by vertical lines) as described under “Experimental Procedures.” The amount of immobilized PKARI corresponded to 5000 res- onance units. Controls with GST alone (C) were also performed. Representa- tives of six similar independent experiments are shown. KD values presented are the mean S.E. from six different experiments.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Binding Assay

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 4. Competition between PKAc and RSK1 for association with PKARI regulates PKA activity. A, PKAc competes with RSK1-(1–317) for binding to PKARI. PKARI (10 pmol) was preincubated with different indicated amounts of PKAc to form the holoenzyme before mixing with glutathione resin pre- bound to GST-RSK1-(1–317) (5 g) for the pulldown assay. GST-RSK1-(1–317) was stained with Coomassie Blue. The panel on the right is quantification of the ratio of the band intensities of PKARI relative to GST-RSK1 from three similar experiments. *, p 0.05; ** p 0.01, Student’s unpaired t test analysis. B, GST full-length RSK1 (GST-FL-RSK1) competes with PKAc for PKARI and decreases the formation of the PKA holoenzyme. PKAc (2 nM final concentration) and increasing indicated concentrations of GST-FL-RSK1 (or GST 100 nM) were added to PKARI (3 nM). After incubation of the mixture for 1 h, formation of the PKA holoenzyme was monitored by measuring PKAc activity as described under “Experimental Procedures.” C, increasing expression of HA-RSK1 (S221A) decreases the association of endogenous PKAc with PKARI. HEK293T cells were transfected with the indicated different amounts of plasmid expressing HA-RSK1 (S221A). Cell lysates were immunoprecipitated (IP) with anti-PKARI antibody. The right hand panel shows the quantification of band intensities of PKAc or HA-RSK1 as a ratio of the band intensities of PKARI from 3 similar experiments. *p 0.05; **, p 0.01. D–E, overexpression of HA-RSK1 (S221A) activates PKA. Experiments were the same as in panel C, except that the cell lysates were probed with anti-phospho-PKA substrate (D) or anti- phospho-BAD Ser-155 antibodies (E). In D, a representative of three similar experi- ments is shown. IB, immunoblot. In E, the right-hand panel shows quantification of band intensities of phospho-BAD-Ser-155 and Erk1/2 from three experiments; *, p 0.05. F, silencing of RSK1 increases the interactions between endogenous PKAc and PKARI. B82L cells were transfected with RSK1-specific siRNA #1 (20 nM) or #2 (40 nM) for 56 h and then serum-starved overnight. The cell lysates were immunoprecipi- tated with anti-PKARI antibody. The right-hand panel shows quantification of band intensities of PKAc as a ratio of PKARI band intensities from three experiments. *, p 0.05, as compared with control. G, silenc- ing of RSK1 decreases phosphorylation of BAD on Ser-155. RSK1 was silenced as in F. Quantified band intensities of phospho-BAD-Ser-155 and Erk1/2 from three experiments are shown on the right. *, p 0.05; **, p 0.01, as compared with control siRNA (Con). WCL, whole cell lysate.

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Activity Assay, Binding Assay, Staining, Concentration Assay, Incubation, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Over Expression, Western Blot, Control, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

doi: 10.1074/jbc.m109.083642

Figure Lengend Snippet: FIGURE 5. PKARI/RSK1 interactions regulate RSK1 activation and apoptosis. A, peptide (Wt-PS) corre- sponding to the pseudosubstrate region of PKARI disrupts the interaction of PKARI and RSK1 and increases RSK1 phosphorylation in unstimulated cells. B82L cells were serum-starved overnight and then were treated with 2 M each of Wt-PS or Mut-PS (for sequences, see the legend to Fig. 2) for 10 min. RSK1 from the cell lysates was immunoprecipitated (IP) with anti-RSK1 antibody. The proteins in the immune complex were detected with Western analysis. The quantitative data using band intensities of the indicated proteins from three exper- iments are shown in the panels on the right. B, Wt-PS, but not Mut-PS, disrupts the association of RSK1 with PKARI, increases the binding of endogenous PKAc to PKARI, and decreases phosphorylation of BAD on Ser-155. Cell lysates were prepared as in A, and PKARI was immunoprecipitated with anti-PKARI antibody. The proteins in the immune complexes or whole cell lysates (WCL) were subjected to Western analysis. Quan- tification of data using band intensities of the indicated proteins (n 3 experiments) are shown in the panels on the right. C, Wt-PS, but not Mut-PS, increases basal RSK1 activity and decreases apoptosis. After overnight deprivation of serum, B82L cells were treated with PKARI peptides Wt-PS or Mut-PS (2 M each) for 10 min followed by stimulation with 50 nM EGF for 10 min. Cell lysates were examined for phosphorylation of Ser-380 on RSK1 and Ser-112 on BAD. The panels on the right represent quantification of band intensities of the indi- cated proteins from three experiments. For the apoptosis assay, after treatments with or without EGF for 10 min, cells were treated with TNF- (20 ng/ml) plus cycloheximide (25 g/ml) (TNF/CHX) for 1 h, and DNA fragmentation was monitored. Data are the mean S.E. of A405 per g of protein (n 3). D, silencing of RSK1 abrogates the ability of Wt-PS to inhibit apoptosis. Procedures were as in C, except that cells were transfected with RSK1 siRNAs for 56 h before experimentation. The inset shows silencing of RSK1 by the two siRNAs. Data are the mean S.E. of A405 per g of protein (n 3).

Article Snippet: The supernatants (500 g of protein except for immunoprecipitation with anti-PKARI antibody, where 1mg protein was used) were incubated for 2 h at 4 °Cwith 0.4 g of anti-RSK1 antibody or 2 g of anti-PKAc antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of PKARI antibody (BD Biosciences) together with 15 l of protein G-conjugated agarose beads.

Techniques: Activation Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Binding Assay, Activity Assay, Apoptosis Assay, Transfection