Journal: PLoS ONE

Article Title: Btk Regulates Macrophage Polarization in Response to Lipopolysaccharide

doi: 10.1371/journal.pone.0085834

Figure Lengend Snippet: (A) WT and Btk −/− BMDMs were treated ex vivo with LPS (100 ng/ml) or IFN-γ (100 U/ml) for 3 hours or 15 minutes, respectively, lysates were prepared and phosphorylated Y701-STAT1 levels determined by Western blot. WT and Btk −/− BMDMs were treated ex vivo with an M1 or M2 polarizing cocktail over the indicated time course, lysates were prepared and tyrosine phosphorylated STAT1 and STAT6 (B), serine phosphorylated AKT (upper panel) and NF-κB p65 (lower panel) (C) iNOS and SHIP-1 (D) levels were determined by Western blot. Results in each case are representative of three independent experiments. Densitometric analysis was performed and graphs represent phosphorylated protein levels relative to unphosphorylated proteins (B-C) or changes in total protein levels relative to β-actin (D) for WT and Btk −/− BMDMs. Student’s paired t test was performed comparing relative expression of phosphorylated or total proteins in Btk −/− BMDMs to WT BMDMs following treatment with polarizing cocktails as indicated. Results shown are mean±SD from three independent experiments. * = p≤0.05.

Article Snippet: Expression of STAT1(Santa Cruz Biotechnology #sc-592), STAT6 (Santa Cruz Biotechnology #sc-621), pY-STAT1 (Cell Signaling #9171), pY-STAT6 (Imgenex #IMG408A), Akt (Cell Signaling #9271), pS-Akt (Cell Signaling #9272), NF-κB p65 (Santa Cruz Biotechnology #sc-372), pS-NF-κB p65 (Cell Signaling #3036S), iNOS (Transduction Laboratories #N39120) and SHIP-1 (Santa Cruz Biotechnology #sc8425), was determined by Western blot as described previously .

Techniques: Ex Vivo, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Btk Regulates Macrophage Polarization in Response to Lipopolysaccharide

doi: 10.1371/journal.pone.0085834

Figure Lengend Snippet: (A) This study has demonstrated that in response to LPS and IFN-γ Btk contributes to M1 polarizing of myeloid cells via promoting the phosphorylation of Akt and subsequently the p65 subunit of NFκB, in addition to enhancing to phosphorylation of STAT1. (B) In the absence of Btk exposure of myeloid cells to LPS and IFN-γ results in the preferential induction of M2 associated genes and preferentially recruitment of M2 cells in vivo . Previous studies in Btk −/− mice have observed increased levels IL-10 systemically following LPS treatment. IL-10 is known to activate STAT3 and there is some evidence to suggest that STAT3 may also play a role in promoting M2 macrophage polarization. Thus in the absence of Btk, in response to M1 polarizing stimuli increased IL-10 production together with reduced phosphorylation of key signaling intermediaries, combined with activation of p50 the inhibitory subunit of NF-κB could potentially account for the observed preferential skew towards an M2 phenotype. Additionally this study has shown that in response to IL-4 and IL-13 Btk −/− cells demonstrate an increased capacity to polarize towards an M2 phenotype as a result of enhanced STAT6 phosphorylation and increased SHIP1 expression.

Article Snippet: Expression of STAT1(Santa Cruz Biotechnology #sc-592), STAT6 (Santa Cruz Biotechnology #sc-621), pY-STAT1 (Cell Signaling #9171), pY-STAT6 (Imgenex #IMG408A), Akt (Cell Signaling #9271), pS-Akt (Cell Signaling #9272), NF-κB p65 (Santa Cruz Biotechnology #sc-372), pS-NF-κB p65 (Cell Signaling #3036S), iNOS (Transduction Laboratories #N39120) and SHIP-1 (Santa Cruz Biotechnology #sc8425), was determined by Western blot as described previously .

Techniques: In Vivo, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

doi: 10.1371/journal.pone.0070891

Figure Lengend Snippet: A , Two potential Akt consensus motifs were identified in N-CoR. The screen capture of the results of the N-CoR putative kinase recognition motif search in the human protein reference database program is shown. Only the sequences that matched to the Akt substrate motifs in the search are presented (upper panel). The Akt substrate motif and it surrounding sequences are highly conserved in human and mouse N-CoR sequences (lower panel). B, Genetic ablation of Akt abrogated N-CoR phosphorylation. Level of N-CoR protein phosphorylated (upper panel, left) at the Akt consensus motif in THP-1 cells treated with AEBSF or Akt siRNA was determined by staining the immunoprecipitated (IP) full length N-CoR protein (lower panel, left) with phospho-Akt substrate (RXRXX pS/pT) antibody. The levels of N-CoR, pAkt (Ser 473) and Akt in crude cellular extracts used in IP assay were determined (right panel). C , Level of N-CoR protein phosphorylated (upper panel, left) at the Akt consensus site in flag-tagged N-CoR protein immunoprecipitated (lower panel, left) from 293T cells transfected with myr-Akt or control plasmid was determined by western blotting with a phospho-Akt substrate (RXRXX pS/pT) specific antibody. An aliquot of crude cell extract was probed with the respective antibodies to determine the levels of flag-N-CoR, pAkt (Ser 473) and Akt (right panel). D, Constitutively active Akt phosphorylates N-CoR in vitro . Affinity purified flag-tagged N-CoR was incubated with purified myr-Akt and the level of phosphorylated N-CoR protein after the incubation was determined by western blotting assay with the phospho-Akt substrate (RXRXX pS/pT) antibody.

Article Snippet: Akt, phospho-Akt (Ser473) and phospho-Akt substrate (RXRXX pS/pT) antibodies were purchased from Cell Signaling Technologies (MA, USA).

Techniques: Staining, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, In Vitro, Affinity Purification, Incubation, Purification

Journal: PLoS ONE

Article Title: Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

doi: 10.1371/journal.pone.0070891

Figure Lengend Snippet: Serine to Alanine substitution at position 1450 completely abrogated the Akt-induced N-CoR phosphorylation. A, Schematic representation of various serine or threonine N-CoR mutant constructs. B, Level of phosphorylated N-CoR (upper panel) in total N-CoR protein immunoprecipitated (lower panel) with Flag antibody from 293T cells transfected with flag-tagged WT or mutant (S1450A and T1925A) N-CoR expression plasmids with or without constitutively active Akt (myr-Akt) was determined with phospho-Akt substrate (RXRXX pS/pT) antibody. To determine the levels of WT and mutant N-CoR proteins, an aliquot of whole cell extract used in the immunoprecipitation assay described above was probed with flag antibody (right panel). Levels of pAkt (Ser 473) and Akt in whole cell extract were probed with the respective antibodies (right panel).

Article Snippet: Akt, phospho-Akt (Ser473) and phospho-Akt substrate (RXRXX pS/pT) antibodies were purchased from Cell Signaling Technologies (MA, USA).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Transfection, Expressing