Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) Image of a COS-7 cell transiently expressing p104-CT-V5, fixed with ice-cold methanol and stained with anti-EB1 (red) and anti-V5 (green); the white rectangle indicates the magnified cytoplasmic region. DNA is stained with DAPI (blue). Scale bar = 10 µm. (B) The N-terminal region of p104 (p104-NT-V5) does not localize to MT plus ends. (C) Mutation of the SKIP motif to SKNN abolishes plus end localization. (D) Image of over-expressed p104-CT-V5 co-localizing with EB1-GFP at bundled MTs in COS-7 cells.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Expressing, Staining, Mutagenesis

Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) Image of TaC12 cell expressing EB1-GFP; the schizont was stained using 1C12 (red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (B) EB1 binding to the schizont does not require MTs: TaC12 cells expressing EB1-myc were treated with 0.1 µg/ml nocodazole for 16 h, collected by mitotic shake-off, fixed with 4% PFA on ice, and stained with anti-myc (green) and anti-tubulin (red) antibodies. The last panel (CONTROL) shows a cell in prometaphase that was not treated with nocodazole, to point out the effect of nocodazole on depolymerizing MTs. DNA is stained with DAPI (blue). Scale bar = 5 µm. (C) Truncation analysis of EB1 binding to the schizont surface. TaC12 cells were transfected with plasmids encoding either full-length GFP-, V5- or myc-tagged EB1, or deletion constructs encoding different domains of EB1 tagged with copGFP.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Expressing, Staining, Binding Assay, Transfection, Construct

Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) COS-7 cell lysates were incubated with Halo-tagged p104-CT-V5, p104-NT-V5 and p104-CT SKNN -V5 linked to resin. The resin was washed with a high salt wash buffer and proteins eluted from the Halolink resin by cleavage with TEV protease. Eluates were subjected to immunoblot analysis using anti-V5 (to reveal p104-NT-V5, p104-CT-V5 and p104-CT SKNN -V5 in the eluates), anti-EB1 (to monitor EB1/p104 interaction) and anti-tubulin (as a control). (B). Lysate from TaC12 cells was subjected to pull-down using GST-EB1 or GST alone as a negative control. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (10% gel) followed by immunoblot analysis using anti-p104 (1C12, upper panels) and anti-TaSP (middle panels). Control samples (right) were treated in the same manner. Ponceau staining (lower panels) shows cleaved-off EB1 (marked by asterisk). (C) Lysates of COS-7 cells expressing full-length EB1-GFP, EB1 125–268 -GFP and EB1 1–133 -GFP were subjected to pull-down analysis using recombinant p104-CT-V5 and p104-CT SKNN -V5 as described above. EB1-GFP and EB1 125–268 -GFP interacted with p104-CT-V5. EB1 1–133 -GFP failed to interact with p104-CT-V5, and p104-CT SKNN -V5 did not interact with any EB1 fragments. The lower panels represent immunoblots probed with anti-GFP to demonstrate the presence of EB1-GFP fragments in pull-down elutions. The top panel shows the same blot reprobed with anti-V5 to demonstrate the presence of bait proteins. EB1 125–268 -GFP is found as a doublet in transfected COS-7 cells, likely due to the presence of a second in-frame translational start codon. In all panels, 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Incubation, Western Blot, Negative Control, Binding Assay, SDS Page, Staining, Expressing, Recombinant, Transfection

Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) TaC12 cells were fixed with ice-cold methanol, and sequentially stained with rat mAb anti-EB1 (green) and anti-p104 (1C12; red). DNA is stained with DAPI (blue). (B) TaC12 cells were synchronized in specific cell cycle stages and stained with anti-EB1 (green) and anti-p104 (1C12, red) as above. Scale bars = 5 µm.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) Immunoblot showing increased p104 phosphorylation during mitosis. 6% SDS-PAGE gels were utilized, allowing multiple forms of p104 phosphorylation to be detected. Mitotic TaC12 cells were obtained by mitotic shake-off and subsequent incubation for 1 h with the proteasomal inhibitor MG132. Lysates were subjected to immunoblot analysis using anti-p104 (1C12). ‘PPase’ indicates that lysate was treated with lambda phosphatase. Increased levels of cyclin B reflect synchronization in mitosis; tubulin was used as a loading control. (B) During mitosis, significant phosphorylation occurs in the vicinity of the SKIP motif. COS-7 cells expressing p104 521–634 -V5 were synchronized in mitosis and lysates analyzed as above. (C) Localization of EB1 at the schizont surface correlates inversely with Cdk1 activity. TaC12 cells were synchronized in a metaphase-like state by mitotic shake-off followed by treatment with the proteasomal inhibitor MG132. Cdk1 inhibition (RO-3306, 10 µM) was carried out for 30 min in the presence of MG132 prior to fixation with ice-cold methanol. Cells were stained sequentially with rat anti-EB1 (green) and anti-p104 (1C12, red). DNA is stained with DAPI (blue). Scale bar = 5 µm. (D). Immunoblot showing altered phosphorylation following Cdk1 inhibition. Mitotic TaC12 cells were obtained as described above. Cdk1 activity was inhibited in mitotic cells by the addition of RO-3306 (10 µM) for 30 minutes (mitotic + RO-3306). Lysates were subjected to immunoblot analysis using anti-p104 (1C12), anti-cyclin B and anti-tubulin. (E). Pull-down analysis reveals that both phosphorylated and unphosphorylated p104 can interact with recombinant EB1. Lysates were prepared from unsynchronized, mitotic and Cdk1-inhibited TaC12 cells. as described, and subjected to pull-down using GST-EB1. EB1 and its potential binding partner(s) was cleaved off GST-EB1 using precision protease and subjected to SDS-PAGE (6% gel) followed by immunoblot analysis using anti-p104. Ponceau staining (lower panel) shows cleaved-off EB1. 1% of the lysate (10 µl of 1 ml) was loaded as input; pull-down lanes represent 10% of eluted protein.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Western Blot, SDS Page, Incubation, Expressing, Activity Assay, Inhibition, Staining, Recombinant, Binding Assay

Journal: PLoS Pathogens

Article Title: Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

doi: 10.1371/journal.ppat.1003346

Figure Lengend Snippet: (A) Images of COS-7 cells expressing either p104-CT-V5 (top panel) or p104-CT SKNN -V5 (lower panel) fused to the mitochondrial targeting sequence of vaccinia virus protein F1L. Mitochondria were visualized using Mitotracker (red) 24 h post transfection prior to fixation in ice-cold methanol and staining with rat anti-EB1 (green) and mouse anti-V5 (white). (B) Image showing 2 transfected cells expressing p104-CT-V5 targeted to mitochondria and one untransfected cell (top); transfected cells were fixed with ice-cold methanol and stained with rat anti-EB1 (green) and mouse anti-V5 (red). DNA is stained with DAPI (blue); Scale bar = 10 µm. Note the presence of MT-associated EB1 in the untransfected cell.

Article Snippet: Primary antibodies used for western blotting and immunofluorescence analysis were mouse mAbs anti-p104 (clone 1C12), anti-V5 (Invitrogen), anti-α-tubulin (clone DM1A, Sigma), anti-cyclin B1 (clone GNS-11, Pharmingen), c-Myc (9E10, Santa Cruz), anti-His (GE Healthcare) rat monoclonal anti-EB1 (clone KT51 Absea), and rabbit polyclonal anti-Turbo GFP (AB514, Evrogen).

Techniques: Expressing, Sequencing, Transfection, Staining

Journal:

Article Title: Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

doi: 10.1091/mbc.02-04-0059

Figure Lengend Snippet: PLD2 localization with different organelles in rat NRK cells. Cells were prepared for immunofluorescence microscopy and incubated with rabbit PLD2 antibody PLD2-27 (A, D, G, and J) and costained with monoclonal antibodies to the ER marker BiP (B); the transferrin receptor (E), a marker of early endosomes and the plasma membrane; and lgp120 (H), a late endosome/lysosomal marker. (K) Cells were stained with caveolin-1, a marker for caveoli; the arrow corresponds to caveolin-1 localized to the plasma membrane. Images were merged to determine overlap between PLD2 (red) and the respective marker proteins (green; C, F, I, and L). Areas of maximal overlap are yellow. (L) Inset, enlargement of the region of overlap between PLD2 and caveolin-1 in the perinuclear Golgi apparatus. The arrow indicates areas where PLD2 puncta (red) either overlap completely with caveolin-1 (green) or are directly adjacent. Images are from projected Z-series. Bars, 10 μm.

Article Snippet: Purified monoclonal anti-rat transferrin receptor antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).

Techniques: Immunofluorescence, Microscopy, Incubation, Marker, Staining