Anti phospho bad

Honokiol (≥ 98% of purity) was purchased from LKT laboratories (Minneapolis, MN). Anti-phospho-EGFR (Y1068), anti-total EGFR, anti-phospho-Akt (S473), anti-total Akt, anti-phospho-ERK (T202/Y204), anti-total ERK, anti-phospho-STAT3 (Y705), anti-total STAT3, anti-cyclin D1, anti-CDK2, anti-CDK4, anti- phospho-Rb (S807/811), anti-p27, anti-caspase 3, anti-phospho-GSK3α/β (S21/9), anti-IκBα, anti-bax, anti-phospho-Bad, anti-β-actin, anti-caspase 8, anti-caspase 9 and goat anti-rabbit IgG secondary antibody were from Cell Signaling Technology (Beverly, MA). Anti-poly (ADP-ribose) polymerase (PARP) and anti-p21 were obtained from Santa Cruz Biotechnology. Erlotinib (99% of purity) was purchased from LC Laboratories (Woburn, MA). NNK (99% of purity) was synthesized as described elsewhere [60 (link)].

Immunoprecipitation was performed to examine the interaction of phospho-Bad and 14-3-3 interaction and Bcl-2 family proteins interaction. Protein samples (200 μg) were precleared with protein A/G agarose beads (Santa Cruz Biotechnology) to eliminate nonspecific binding proteins. They were mixed with following antibodies: anti-14-3-3, anti-Bad, and anti-Bax antibody (Santa Cruz Biotechnology) and incubated for overnight at 4°C with mild shaking. Complexes were precipitated with protein A/G agarose beads for 2 h at 4°C, washed with radioimmunoprecipitation assay buffer (Sigma Aldrich) with PMSF, and centrifuged at 10,000 g for 1 min. Supernatants were removed and remnant were boiled with sample buffer. They were loaded on 10% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to PVDF membrane. Membrane were rinsed with TBST and reacted with following antibodies: anti-phospho-Bad (1:1,000, diluted with TBST, Cell Signaling Technology), anti-Bcl-2, and anti-Bcl-xL antibody (1:1,000, diluted with TBST, Santa Cruz Biotechnology). They were washed with TBST and continuously performed as above described method in Western blot analysis.