k562 nuclear cell extracts  (Santa Cruz Biotechnology)

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: 1q21.1 deletion and a rare functional polymorphism in siblings with thrombocytopenia-absent radius–like phenotypes

doi: 10.1101/mcs.a004564

Figure Lengend Snippet: Electromobility shift assays (EMSAs) suggest that rs61746197 A > G is a functional SNP. ( A ) Ten micrograms of unstimulated nuclear extract from K562 cells was incubated with reference (Ref, R) or alternative (Alt, A) probe as indicated. Cold Ref probe titrates signal away from Alt at a lower stoichiometric ratio than the converse (arrow and compare lanes 11 – 13 with 6 – 8 ). ( B ) Unstimulated extract ( left panel, U) exhibits a markedly stronger baseline shift than TPA stimulated extract ( right panel, S). Unstimulated nuclear extract (U) when incubated with labeled alternative probe (A) produces a novel secondary shift not observed with either the reference probe (R) or the TPA stimulated extract (S). Addition of RelA–p65 antibody abrogates this secondary shift (arrow).

Article Snippet: EMSAs were performed using TPA-stimulated and unstimulated nuclear extracts of K562 cells (Active Motif).

Techniques: Functional Assay, Incubation, Labeling

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 1. VASP and Zyxin modulate apoptosis-related proteins. (A–D) Evaluation of VASP and Zyxin silencing in K562 cells transduced with lentivirus-mediated shRNA control and lentivirus mediated shRNA targeting VASP and Zyxin by quantitative RT-PCR analysis (A, C) and Western blotting analysis (B, D), respectively. (E–G) Western blotting analysis was used for quantification of protein expression and activity. Respective total protein or Actin was used as a control to ensuring equal sample loading; the antibodies used for immunoblotting (IB) are indicated.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: Transduction, shRNA, Control, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 3. VASP and Zyxin silencing does not modulate proliferation and clonogenicity. (A) and (B) Cell proliferation/viability was determined by MTT assay of incubation of shControl or shVASP or shZyxin K562 cells, respectively, treated or not with imatinib mesylate (0.1; 0.5 or 1 μM) after 48 h and normalized by untreated shControl cells. Values are expressed in per- centage, and normalized to the shControl value set as 100%. Results are shown as mean ± SD of at least three independent experiments. The P values are indicated in the figure; two-way ANOVA test and Bonferroni post-test (C) and (D) shControl or shVASP or shZyxin cells were cultured in methyl cellulose and colonies were detected by MTT after 8 days of incubation with or without treated imatinib mesylate (0.1; 0.5 or 1 μM) and were normalized by untreated shControl cells. Values are expressed in percentage, and normalized to the shControl value set as 100%. Colony images are representative of one experiment and the bar graphs are shown as mean ± SD of at least three independent experiments.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: MTT Assay, Incubation, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Imatinib restores VASP activity and its interaction with Zyxin in BCR-ABL leukemic cells.

doi: 10.1016/j.bbamcr.2014.11.008

Figure Lengend Snippet: Fig. 4. Imatinib treatment enhances VASP and Zyxin interaction, increases VASP phosphorylation at serine 157 and reduces association of VASP and BCR–ABL. (A) The same lysates from K562 cells untreated and treated with Imatinib (1 μM) were immunoprecipitated (IP) with anti-Zyxin antibody and immunoblotted (IB) with anti-Zyxin and anti-VASP antibodies. Total protein extract (input) and isotype IgG antibody were used as controls.(B) Western-blotting using anti-p-VASP ser157, anti-VASP and Actin antibodies in K562 cells treated with Imatinib (1 μM) for 3, 6, 9 and 12 h. (C) Endogenous VASP were co-immunoprecipitated with BCR–ABL. Total extracts from K562 cells treated with Imatinib (1 μM) were submitted to immuno- precipitation with an anti-VASP antibody followed by western blot analysis using anti-ABL and anti-VASP antibody. Total protein extract (input) and isotype IgG antibody were used as controls. (D) Western blotting showed phospho-VASP ser 157, VASP, phospho-Zyxin ser 142 and Zyxin in K562 cells, one healthy donor, one CML patient at diagnosis, one responding and two resistant to Imatinib. Actin staining indicates the amount of protein loaded. All images in Fig. 4E belong to the same western blotting and the membrane was stripped and reprobed with different antibodies. Actin was used as a control to ensuring equal sample loading; the antibodies used for immunoblotting (IB) are indicated.

Article Snippet: Briefly, 500 μg of total K562 cell extracts was incubated overnight with 20 μL anti-VASP antibody (Santa Cruz Biotechnology) or with normal goat immunoglobulin (IgG) as a negative control.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Biomarker Discovery, Staining, Membrane, Control