rabbit polyclonal anti-mklp1 (n-19)  (Santa Cruz Biotechnology)

Journal: Cell Cycle

Article Title: A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

doi: 10.4161/15384101.2014.942204

Figure Lengend Snippet: The C-terminal region of HMG20b mediates localization to midbody. ( A ) Localization of HMG20b during cell cycle. Confocal images of HeLa cells expressing GFP-HMG20b are shown for each cell cycle phase. Enlarged image of midbody is shown as an inset . Scale bar is 5 μm. ( B ) Western blot showing inducible expression of GFP-HMG20b fragments. β-Actin was probed as a loading control. ( C ) Confocal images of HeLa cells expressing GFP-HMG20b fragments (green). MKLP1(red) was immunostained as a midbody marker. Enlarged images of midbody are shown as insets. Scale bar is 5 μm. ( D ) GFP intensity at midbody normalized to the intensity within total cell area for each GFP-HMG20b fragments. A representative scatter dot plot from 3 independent experiments is shown with mean ± s.e.m. Twenty-five cells were analyzed in each experiment. Statistical significance was confirmed for the indicated pair-wise comparisons using Dunnett's multiple comparison test (***: P < 0.001).

Article Snippet: MKLP1 was stained with anti-MKLP1 rabbit polyclonal antibody (Santa Cruz Biotechnology (N-19, sc-867), 1:1,000)).

Techniques: Expressing, Western Blot, Control, Marker, Comparison

Journal: Cell Cycle

Article Title: A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

doi: 10.4161/15384101.2014.942204

Figure Lengend Snippet: Endogenous HMG20b localizes to the cytokinetic midbody. ( A ) Western blot analysis of control and HMG20b siRNA-treated HeLa cell extracts using HMG20b monoclonal antibody 1F6. Purified His-HMG20b protein was used as a positive control and β-Actin was probed as a loading control. ( B ) Confocal images of control and HMG20b siRNA-treated HeLa cells stained with anti-HMG20b monoclonal antibody 1F6 (green). MKLP1 (red) was co-stained as a midbody marker. Enlarged images of midbody are shown as insets. Scale bar is 5 μm. ( C ) Mean intensity of HMG20b staining at midbody. Results from 50 cells per sample are shown with mean ± s.e.m. (***: P < 0.001, unpaired t test)

Article Snippet: MKLP1 was stained with anti-MKLP1 rabbit polyclonal antibody (Santa Cruz Biotechnology (N-19, sc-867), 1:1,000)).

Techniques: Western Blot, Control, Purification, Positive Control, Staining, Marker

Journal: Cell Cycle

Article Title: A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

doi: 10.4161/15384101.2014.942204

Figure Lengend Snippet: A cancer-associated mutation in the C-terminal region of HMG20b disrupts cytokinesis in a dominant manner. ( A ) GST-pull down assay. Flag-HMG20b mutants expressed in 293T cells were incubated with GST-BRCA2 fragment containing BRC5. Bound proteins were detected by western blotting using anti Flag antibody. Ponceau S- stained membrane is included in the lower panel to show that equal amount of GST fusion proteins was used for each sample. ( B ) Confocal images of HeLa cells expressing wild type or A247P mutant of GFP-HMG20b (green). MKLP1 (red) was immunostained as a midbody marker. Enlarged images of midbody are shown as insets. Scale bar is 5 μm. ( C ) Localization of wild type and A247P mutant of GFP-HMG20b at midbody. A representative scatter dot plot of GFP intensity ratio from 3 independent experiments is shown with mean ± s.e.m. Twenty-five cells were analyzed in each experiment (***: P < 0.001, unpaired t test). ( D ) Clone (A247P-5) expressing Flag-HMG20b with A247P mutation was analyzed for multinucleation frequency after HMG20b siRNA (Q1) and/or Doxycycline (Dox) treatment. Means ± s.e.m. from 3 experiments are shown and >500 cells were analyzed per each sample. Statistical significance was confirmed for the indicated pairwise comparisons using Bonferroni's multiple comparison test (**: 0.001 < P < 0.01, *: 0.01 < P < 0.05, ns: P > 0.05). ( E ) Multinucleation frequency measured at 72 hours after HeLa cells were transiently transfected with Flag-HMG20b constructs . Means ± s.e.m. from 4 independent experiments (2 experiments for the vector control) are shown. Transfected cells were identified by anti-Flag immunostaining and >200 transfected cells were analyzed per each sample. Wild type and A247P mutant of HMG20b are compared with the vector control by Dunnett's multiple comparison test (***: P < 0.001, ns: P > 0.05).

Article Snippet: MKLP1 was stained with anti-MKLP1 rabbit polyclonal antibody (Santa Cruz Biotechnology (N-19, sc-867), 1:1,000)).

Techniques: Mutagenesis, Pull Down Assay, Incubation, Western Blot, Staining, Membrane, Expressing, Marker, Comparison, Transfection, Construct, Plasmid Preparation, Control, Immunostaining