Ecl plus chemiluminescence reagent

Western blot analysis was performed as described [30 (link)]. Fifteen μg total protein lysate was separated on 10% SDS-PAGE. Proteins were then transferred to a PVDF membrane (Amersham Biosciences, Piscataway, NJ). The membrane was blocked, incubated with primary antibodies overnight and secondary antibody for 1 h at room temperature. The membrane was developed with ECL Plus chemiluminescence reagent (Amersham Biosciences). The primary antibodies used in this study were against actin (Santa Cruz, CA), peripherin (Santa Cruz, CA), cytokeratin-8 (Abcam, MA), aldose reductase (Abcam, MA), alpha-enolase (Santa Cruz, CA), Grp75 (Cell Signaling Technology, MA), phosphoglycerate mutase (Santa Cruz, CA), F1 ATPase (Santa Cruz, CA), SCO2 (Santa Cruz, CA) and OGDH (Santa Cruz, CA).

SDS-PAGE and Western blot analyses were done as described with slight modifications [22 (link)]. Briefly, 24 hours posttreatment, cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with freshly added 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride and Protease Inhibitor Cocktail (Santa Cruz, CA) and loaded onto 10% polyacrylamide gel. Proteins were then transferred to microporous polyvinylidene difluoride (PVDF) membrane (Milipore). Membranes were incubated in 5% BSA (Sigma) blocking buffer for 1 h at room temperature. Incubations with primary antibody were carried out overnight at 4°C. Immunoblotting was performed with rabbit anti-Bcl-2, anti-Bcl-xl, and anti-Bax antibodies (1 : 200) (Cell Signaling Technology, Danvers, MA). Membranes were washed 3 times (10 min each) in Tween buffer before incubating with HRP-conjugated goat anti-mouse or rabbit secondary antibodies. To remove excess antibodies, membranes were washed 4 times before HRP activities were detected using ECL Plus Chemiluminescence Reagent (Amersham, Chalfont, UK) according to the protocol supplied with the kit.

The extracts are heated in a boiling water bath for 5 min and equal amounts of protein (40 μg) were separated by 10 % polyacrylamide gel. Proteins were then transferred to microporous polyvinylidene difluoride (PVDF) membrane (Milipore). Membranes were incubated in 5 % BSA (Sigma) blocking buffer for 1 h at room temperature. Incubations with primary antibody were carried out overnight at 4 °C. Immunoblotting was performed with the following antibodies: anti-FOXO3a, anti-p-FOXO3a (Ser-253), anti-p21Cip1/waf1, anti- p27Kip1, anti-Bim, anti-cyclinD1, anti-cyclinE, anti-Akt (total), anti-p-Akt (Thr-308), anti-p-PI3K, anti-total-PI3K, anti-GAPDH, anti-LaminB1 (1:1000) (Cell Signaling Technology, Danvers, MA) and mouse anti-β-actin (1:10000) (Sigma) antibodies overnight at 4 °C. The next day, the membranes were washed three times for 10 min in TBS-T and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h. To remove excess antibodies, membranes were washed 4 times before HRP activities were detected using ECL Plus Chemiluminescence Reagent (Amersham, Chalfont, UK) according to the protocol supplied with the kit. Finally the quantification was done by ImageJ software (NIH, Bethesda, MD).