Anti bcl xs l

The following antibodies and chemicals were purchased: anti- Cullin-4B, anti-FLAG (M2), and anti-α-tubulin antibodies, mouse IgG-agarose, monoclonal anti-HA-agarose, and anti-FLAG M2 affinity gel from Sigma-Aldrich Co. (St. Louis, MO); anti-caspase-3, anti-caspase-9, anti-cleaved caspase-3, and anti-cleaved caspase-7 antibodies from Cell Signaling Technology (Beverly, MA); anti-Apaf-1 antibody from Millipore (Temecula, CA) and Abcam (Cambridge, United Kingdom); anti-multi ubiquitin antibody (FK2) from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan); anti-p62 (C-terminal) antibody (specific to amino acids 421–440 of human p62 protein) from PROGEN Biotechnik GmbH (Heidelberg, Germany); anti-HA (HA.11 and Y-11) and anti-myc antibodies from Covance (Richmond, CA) and Santa Cruz Biotechnology (Santa Cruz, CA); and anti-ATF4, anti-REST, and anti-Bcl-xS/L antibodies from Santa Cruz Biotechnology. HRP-conjugated anti-rat, anti-mouse, anti-rabbit, and anti-guinea pig IgG (H + L) antibodies were purchased from Southern Biotech (Birmingham, AL); MG132 from Calbiochem (La Jolla, CA); etoposide, E64d, pepstatin A, and cycloheximide from Sigma-Aldrich Co. All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), Kanto Chemical (Tokyo, Japan), and Sigma-Aldrich Co.

Transfected cells were washed and homogenized in a protein lysis buffer (RIPA buffer). Protein concentrations were quantified using BCA Protein Assay Kit (Pierce). 10 μg of each sample were denaturized at 95^°C for 10 min and further processed using the NuPAGE SDSPAGE Gel System (Invitrogen). Proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL, GE Healthcare), which was treated afterwards with 5% skimmed milk powder in TBS Tween 0.1%. Primary antibodies were applied to the membrane according to manufactures’ protocol: anti-p-AKT Ser473 (Cell Signaling), anti-AKT (Cell Signaling), anti-BCL-X S/L (Santa Cruz Biotechnology), anti-cFLIP S/L (Adipogen) anti-COPB2 (novus Biologicals), anti-p-ERK1/2 Thr202/Tyr204 (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-GAPDH (Cell Signaling), anti-KRAS (Santa Cruz Biotechnology), anti-LC3 (Cell Signaling), anti-pSTAT3 (Tyr705) (Cell Signaling), anti-STAT3 (Cell Signaling), anti-PARP (Cell Signaling), anti-XIAP (BD Biosciences). The membrane was washed and incubated with the appropriate horseradish peroxidase-conjugated antiMouse or anti-Rabbit secondary antibodies (Cell Signaling). The chemiluminiscent reaction was initialized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and detected by G:Box Chemi XT4 (Syngene) (Figures 4, 5, 6).