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: PLoS ONE

Article Title: Circumvention of Mcl-1-Dependent Drug Resistance by Simultaneous Chk1 and MEK1/2 Inhibition in Human Multiple Myeloma Cells

doi: 10.1371/journal.pone.0089064

Figure Lengend Snippet: ( A ) and ( B ) U266 ( A ) and H929 ( B ) cells were exposed to 500 nM CEP3891±PD184352 (7.5 µM for U266; 2.5 µM for H929) for 42 h; ( C ) and ( D ) U266 cells over-expressing Mcl-1 and their EV controls were treated as described for U266 in panel 2A. After treatment, cells were lysed in 1% CHAPS buffer and immunoprecipitated (IP) using anti-Mcl-1 ( C ) or anti-Bak ( D ) antibodies, followed by Western blot (WB) analysis using anti-Bim, anti-Bak, or anti-Mcl-1 antibodies as indicated. WCL were loaded to monitor Bim levels. ( E ) and ( F ) Alternatively, following 24 h treatment, IP was performed to monitor conformational change of Bax and Bak using anti-Bax 6A7 or anti-Bak Ab-1 (for IP), and anti-Bax or anti-Bak (for WB) in parental U266 cells ( E ) and their counterparts ectopically expressing Mcl-1 ( F ). For all IP assays, IPs without cell lysate (-lysate) and/or with IgG (instead of primary antibodies) were carried out as controls; 200 µg protein per condition were employed for IP; IgG levels are shown to ensure equal loading of IP antibodies.

Article Snippet: Cells were lysed in CHAPS buffer and 200 μg of protein per condition were immunoprecipitated with 1 μg anti-Mcl-1 (Santa Cruz Biotechnology or BD Biosciences), anti-Bak, or anti-Bim (Santa Cruz Biotechnology), followed by Dynabeads (Dynal, Oslo, Norway).

Techniques: Expressing, Immunoprecipitation, Western Blot