Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 1. Western immunoblot of (a) HeLa cell lysate and (b) KIF20B-overexpressing HEK293T cell lysate (see Fig. 4) with a monoclonal to the *97-kDa Golgi complex protein golgin 97(21) (lane 1) and monoclonal 10C7 anti- KIF20B (lane 2). KIF20B is identified as a *2325-kDa protein (arrows). Conditions: (a) 50 mg of Hela cell lysate loaded on gel and probed with monoclonal 10C7 diluted 1:4. (b) 80mg of KIF20B-overexpressing HEK293T cell lysate loaded on gel and probed with monoclonal 10C7 diluted 1:4.

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques: Western Blot

Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 2. Indirect immunofluorescence of 10C7 monoclonal antibody to KIF20B on HEp-2 cells. Nuclei (a, b asterisks) of *20% of cells were stained, with intense staining of the intracellular bridge and midbody region (arrows in b–d). Faint speckling of cytoplasm was consistently observed and the interchromatin mass of late M-phase cells (b) is also stained (spinning disk confocal, original magnification 600 · ).

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques: Staining

Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 3. Monoclonal 10C7 directed to KIF20B (1:10 diluted in PBS) reacts with (a) nuclei and the intercellular bridge (arrows) of human leptomeningeal pericyte cells (cells fixed and permeabilized with 4% PFA/0.2% Triton X-100); (b) granular layer (GL) and Purkinje cells (arrows) of monkey cerebellum; and (c) nuclei in monkey testis and (d) ovary tissues. Goat anti- mouse IgG Alexa488 diluted at 1:500 in PBS was used as the secondary antibody. PBS, phosphate-buffered saline.

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques: Saline

Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 4. HEK293T cells were transfected with full-length human KIF20B cDNA (transfected) or exposed to the trans- fection carrier only (mock), and after incubation, to allow overexpression of KIF20B cDNA (OriGene Technologies, Inc.), they were fixed and permeabilized with 3% PFA/0.2% Triton x-100 and stained with monoclonal 10C7 anti-KIF20B (diluted at 1:10 in PBS) and DAPI (visualize cell nuclei). KIF20B is overexpressed in transfected cells compared with mock treated cells and the typical, variable, speckled cell staining was observed in *50% of cells (merge).

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques: Transfection, Incubation, Over Expression, Staining

Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 5. Monoclonal 10C7 anti-KIF20B colocalization (diluted at 1:10 in PBS) with rabbit anti-CENP-F (mito- sin, diluted at 1:1000 in PBS) on commercially available HEp-2 cells (ImmunoConcepts). At telophase, KIF20B is concentrated within the intracellular bridge at either side of the midbody (arrows). CENP-F, centromere protein F.

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques:

Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy

Article Title: A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

doi: 10.1089/mab.2019.0016

Figure Lengend Snippet: FIG. 6. MPP1/KIF20B is characterized by extensive coiled-coil regions except for two regions (amino acids 1–200 and approximately 275–560) at the carboxyl terminus and then a similar, but smaller, region at the amino terminus (aa 1575– 1800). Note that the epitopes reactive with 10C7 (amino acids 1772–1787, Uniprot: Q96Q89) and the human anti-KIF20B serum samples (amino acids 548–560; 1691–1706; carboxy terminus) are not localized to coiled-coil regions of KIF20B. MPP1, M-phase phosphoprotein 1.

Article Snippet: Transfected and nontransfected cell lysates HEK293T cells (American Type Culture Collection, Cedarlane, Burlington, ON, Canada) were seeded in culture plates (Nunc UpCell Surface 10 cm; Thermo Fisher Scientific, Waltham, MA) and incubated for 1 day to enhance attachment before transfection with the full-length human KIF20B cDNA (NM_016195) tagged with green fluorescent protein (GFP) obtained from Origene (Catalog No.: RG215061; Rockville, MD).

Techniques:

Journal: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: Deletion in the DKC1 3′ UTR segregates with disease phenotype. A, Location of the 1104 bp deletion on chromosome X, encompassing part of the DKC1 3′ UTR and the intergenic region between genes DKC1 and MPP1 . B, Magnification of the deleted region, showing the location of polyadenylation signals (PolyA; confirmed in Figure ) and primers used for PCR and sequencing (A‐fwd, B‐rev, C‐seq). C, Sanger sequencing (using primer C‐seq) of the PCR product from primers A‐fwd and B‐rev, in the proband (V‐2) and his father (IV‐2). Black arrow marks the 5′ end of the deletion. D, Agarose gel of products of PCR across the deletion in 12 family members. The proband is indicated in blue; shown in red are female family members with signs of disease. The sizes of the wild‐type (WT) or deleted (ΔUTR) PCR products are shown on the right. Note that DKC1 is on the X chromosome, so male individuals (III‐2, IV‐2, V‐2) carry a single allele

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Sequencing, Agarose Gel Electrophoresis

Journal: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: Short telomeres correlate with presence of the DKC1 deletion. Telomere lengths of 12 family members, measured by qPCR (A) or TRF Southern blot analysis (B). The proband is indicated in blue; shown in red are heterozygous female family members. Curves in (A) represent the indicated percentiles of telomere lengths in ∼240 healthy individuals. Mean TRF lengths in kb are indicated below each lane of the Southern blot in (B)

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Southern Blot

Journal: EJHaem

Article Title: A novel cause of DKC1 ‐related bone marrow failure: Partial deletion of the 3′ untranslated region

doi: 10.1002/jha2.165

Figure Lengend Snippet: The proband expresses very low levels of DKC1 mRNA and telomerase RNA. A, RT‐PCR of DKC1 transcripts in peripheral blood cells of the proband (V‐2) and his immediate family members, and RNA from HeLa cells or an unrelated individual (Ctrl) as wild‐type controls. −RT: negative control in the absence of reverse transcriptase. B, A second round of nested PCR was performed on the products from (A), using an internal primer. C, Levels of DKC1 mRNA, measured by quantitative real‐time RT‐PCR (RT‐qPCR) using random hexamers for cDNA transcription in the proband (V‐2) and his immediate family members. Female relatives heterozygous for the deletion are labeled in red. Data shown as mean ± SEM of expression levels relative to individual IV‐2; **** P < .0001, as determined by two‐way ANOVA followed by Tukey's multiple comparison tests; n = 3‐4 independent RT reactions from each of two blood samples. D, Levels of the telomerase RNA subunit, hTR, measured by RT‐qPCR using random hexamers for cDNA transcription in the proband (V‐2) and his immediate family members. Female relatives heterozygous for the deletion are labeled in red. Data shown as mean ± SEM of expression levels relative to individual IV‐2; **** P < .0001, as determined by two‐way ANOVA followed by Tukey's multiple comparison tests; n = 5 independent RT reactions from each of two blood samples

Article Snippet: A portion (2.5%) of the cDNA was used for real‐time PCR with Platinum SYBR Green qPCR SuperMix‐UDG (Invitrogen) and 400 nM primers DKFA and DKRA for DKC1 , hTR‐F and hTR‐R for TERC , or a qSTAR qPCR primer pair for MPP1 (OriGene Technologies; Table S1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Reverse Transcription, Nested PCR, Quantitative RT-PCR, Labeling, Expressing, Comparison