Journal: Journal of Biological Chemistry

Article Title: Translocation of Full-length Bid to Mitochondria during Anoikis

doi: 10.1074/jbc.m313375200

Figure Lengend Snippet: FIG. 6. Bid translocation and anoikis induction do not require interactions with other Bcl-2 family proteins. a, adherent or detached cells (poly-HEMA) were separated into soluble (Sol.) or membrane fractions. The membrane fraction was then extracted in CHAPS. The CHAPS-insoluble material (Insol.) was solubilized in SDS-PAGE sample buffer. Equal amounts of each were separated by SDS-PAGE. The fractions were immunoblotted for Bid and Bax. As we have previously shown, following 4 h of detachment, the majority of Bax is in the CHAPS-insoluble fraction. Conversely, all the membrane associated Bid is extracted by CHAPS. b, membrane fractions from adherent cells, or cells detached for 15 min or 1 h, were separated by S100 gel filtration. Every third fraction collected was separated by SDS-PAGE and immunoblotted for Bid, Bax, Bcl-XL, and Bak. Bid is always isolated as a monomer. Other Bcl-2 family proteins are always associated with high molecular weight complexes. Only the 15-min samples are shown for Bad and Bak. No difference was seen for those molecules at any time point. Approximate sizes

Article Snippet: Cell Fractionation and Chromatography—For gel filtration of CHAPS-extracted membrane fractions, 5 mg of protein in a volume of 0.5 ml was loaded onto a Sephacryl S100-HR column (1.5 25 cm; Amersham Biosciences; equilibrated in 10 mM HEPES-Cl, pH 7.6, 150 mM NaCl, 4 mM CHAPS).

Techniques: Translocation Assay, Membrane, SDS Page, Filtration, Isolation, High Molecular Weight

Journal: Cancer Research

Article Title: Mcl-1 Phosphorylation Defines ABT-737 Resistance That Can Be Overcome by Increased NOXA Expression in Leukemic B cells

doi: 10.1158/0008-5472.can-11-4106

Figure Lengend Snippet: Figure 1. Acquired resistance development in leukemic cells. A, parental and ABT-737-resistant (ABT-R) Nalm-6 and Reh cells were treated with ABT-737 for 24 hours. Cell viability is shown as a percentage of Annexin V–FITC/ propidium iodide negative, relative to control cells treated with DMSO, as determined by flow cytometric analyses. SD is indicated as error bars (n ¼ 3). B, cells were treated with ABT-737 for 24 hours with 1,000 nmol/L (Nalm-6) or 500 nmol/L (Reh) ABT-737, lysedwith 1%CHAPS lysis buffer, and 1 mg protein was immunoprecipitated for 6A7-specific Bax and immunoblotted for total Bax. C, whole-cell lysates were analyzed by immunoblotting for expression of Bcl-2 family proteins and b-actin by immunoblotting with the indicated primary antibodies. D, parental and ABT-R Nalm-6 cells were treated with ABT-737 (1,000 nmol/L for 1–3 hours) and expressions of Bim, USP 9X, Mcl-1, PARP1, and b-actin was determined by immunoblotting. The results in B to D are representative of 3 independent experiments. IP, immunoprecipitation.

Article Snippet: For Bax activation, cells were lysed with 1% CHAPS lysis buffer for 1 hour at 4 C and active Bax was immunoprecipitated with 6A7 (BD Biosciences) and probed with N20 (Santa Cruz Biotechnology) antibodies.

Techniques: Control, Lysis, Immunoprecipitation, Western Blot, Expressing