Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release

doi: 10.1371/journal.pntd.0005279

Figure Lengend Snippet: A: The percentage of Mf that had at least one PMN adhered to their surface or indirectly fastened by extracellular DNA at 24 hours post-experimental set up (n = 3). Orange and red bars represent 5% autologous serum and 25% autologous serum treated wells respectively. The autologous serum was either untreated (no peptide control), treated with 100μM inactive compstatin analogue (control peptide) or treated with 100μM active compstatin. Error bars represent standard error of the mean. B: The percentage of Mf that survived 5 days in the presence of both PMNs and PBMCs after treatment of serum with control peptide or compstatin (n = 3). C: PMNs were incubated with blocking antibodies for 2 hours prior to incubation with Mf for 5 days. PMN-mediated killing occurred to the same extent in control (no antibody) wells and those treated with anti-ICAM-1 and anti-Cd11b (p = <0.0001). No significant differences were observed between PMN incubated with Mf with or without the anti-ICAM-1 or anti-Cd11b (n = 3).

Article Snippet: For the compstatin inhibition studies, autologous serum was pretreated with either 100μM compstatin (Tocris Bioscience, Avonmouth, Bristol, U.K.) or 100μM compstatin control peptide (Tocris Bioscience, Avonmouth, Bristol, U.K.) for 30 min at 37°C.

Techniques: Control, Incubation, Blocking Assay