Journal: Nucleic Acids Research

Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

doi: 10.1093/nar/gkm1091

Figure Lengend Snippet: Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

Article Snippet: Cell lysates were incubated at 4°C for 1 h with 20 μl of glutathione affinity Sepharose (50% slurry, Amersham Pharmacia Biotech) for GST-PAP.

Techniques: In Vitro, Transfection, Incubation, SDS Page, Autoradiography, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

doi: 10.1093/nar/gkm1091

Figure Lengend Snippet: Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

Article Snippet: Cell lysates were incubated at 4°C for 1 h with 20 μl of glutathione affinity Sepharose (50% slurry, Amersham Pharmacia Biotech) for GST-PAP.

Techniques: Activity Assay, Transfection, Papanicolaou Stain, In Vitro, Labeling, Purification, Thin Layer Chromatography

Journal: Frontiers in Immunology

Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

doi: 10.3389/fimmu.2018.02852

Figure Lengend Snippet: Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).

Article Snippet: GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions.

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Western Blot, Incubation

Journal: Scientific Reports

Article Title: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology

doi: 10.1038/srep25411

Figure Lengend Snippet: The CRIB motifs in mammalian CRN7 and their impact on the rescue activity of the protein. ( a ) Sequence alignment of human CRN7-CRIB domains (NT and CT) with CRIB domains from human Coronin 1C and D. discoideum CRN7. The CRIB consensus shown on top. Similar amino acids boxed in yellow. Highly conserved amino acids marked in red. (b) In vitro binding assay for full length GFP-CRN7WT with Rac1 and Cdc42 GTPases in their CA/Q61L and DN/T17N forms. Glutathione-Sepharose beads coated with GST alone and GST fusions pre-loaded with GDP (DN) or GTPγS (CA) and incubated with lysates from HEK293T cells expressing GFP-CRN7. PonceauS staining shows the GST fusion proteins. GFP-CRN7 detected with GFP-specific mAb K3-184-2. (c) Quantification of GFP-CRN7 bound to Rac and Cdc42 proteins (CA and DN) using ImageJ. Input set at 100% (3 independent experiments; **P

Article Snippet: GST-Cdc42 or CRN7 mutants were expressed in E. coli BL21 and purified from the soluble fraction using Glutathione Sepharose affinity columns (GE Healthcare).

Techniques: Activity Assay, Sequencing, In Vitro, Binding Assay, Incubation, Expressing, Staining

Journal: Scientific Reports

Article Title: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology

doi: 10.1038/srep25411

Figure Lengend Snippet: Effect of CRN7-CRIB motif mutations on Cdc42 binding and rescue potential of the proteins. ( a ) Conserved residues in the N- and C-terminal CRIB domains boxed in yellow were mutated to alanine marked in red. The GFP tag is at the N-terminus. ( b ) Binding assay for GFP-CRN7 WT and CRIB mutants (Mut1 and 2, left panel; Mut3 and 4, right panel) with Cdc42 GTPase (CA and DN). Glutathione-Sepharose beads coated with GST fusions, pre-loaded with GDP (DN) or GTPγS (CA) and incubated with lysates from HEK293T cells expressing the CRN7 WT or CRIB mutants. PonceauS staining shows the GST fusion proteins. Probing was with mAb K3-184-2. (c) Bar graph showing quantification of GFP-CRN7 WT and CRIB mutants bound to Cdc42 CA and DN using ImageJ. Input set at 100% (3 independent experiments; **P

Article Snippet: GST-Cdc42 or CRN7 mutants were expressed in E. coli BL21 and purified from the soluble fraction using Glutathione Sepharose affinity columns (GE Healthcare).

Techniques: Binding Assay, Incubation, Expressing, Staining