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: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate transforming capability and result in hyperactivation of the JAK/STAT pathway. (A) Growth curves of Ba/F3 cells expressing JAK1 A634D and JAK1 R629_S632delinsSA cultured in the absence of IL-3 (graph is representative of 3 independent experiments). (B) Immunoblot analysis of Ba/F3 parental cells, Ba/F3 cells expressing JAK1 wild-type, JAK1 A634D, and JAK1 R629_S632delinsSA cultured in the presence of IL-3 and IL-3–independent Ba/F3 cells expressing JAK1 A634D, and JAK1 R629_S632delinsSA. (C) Immunoblot analysis of HEK293 cells expressing JAK1 wild type, JAK1 A634D, and JAK1 R629_S632delinsSA. p-, phosphorylated; WT, wild-type.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate sensitivity to JAK inhibitors. (A) Representative graphs of the dose-response curves (72-hour sensitivity) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (upper panels). Graphs showing mean IC50 (lower left panel) and area under the dose-response curve (AUC; lower right panel) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (3 independent experiments). (B) Immunoblot analysis of IL-3–independent Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA treated with vehicle or 50 nM or 100 nM of ruxolitinib for 4 hours (n = 2 replicates). (C) Sensitivity of peripheral blood and bone marrow specimens from the patient with JAK1 R629_S632delinsSA to JAK inhibitors, represented by IC50, in comparison with control samples obtained from healthy donors (HD) and specimens from a larger cohort of patients with MPNs) (the horizontal lines denote median IC50 of HD and MPN specimens). ****P < .0001, ***P < .001, 1-way analysis of variance. p-, phosphorylated.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Western Blot, Comparison, Control