22 The membranes were blocked with a solution of 1% bovine serum albumin, incubated with the indicated antibodies (see figure legends), and then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. Monoclonal anti-KDEL antibody was from CalBiochem (San Diego, CA). Anti-phosphorylated α-subunit of eukaryotic translational initiation factor 2 (eIF2α), and anti-eIF2α antibodies were from Oncogene (Boston, MA). Rabbit polyclonal anti-LC3 was from Novus VX-809 mw Biologicals, Inc. (Littleton, CO). After a 2-hour treatment of McA-RH7777 cells

or primary rat hepatocytes with 5 mM GLS or 5 μg/mL TM, the cells were preincubated in methionine/cysteine-free minimum essential medium Selleckchem Alvelestat with 5 mM GLS or 5 μg/mL TM at 37°C for 1 hour, followed by pulse-labeling with 100 μCi/mL [35S]methionine for 2 hours in the presence or absence of 1 mM PBA (see figure legends). Following the pulse, the medium was harvested for immunoprecipitation of secreted apoB100 or albumin. The cells were lysed using 500

μL solubilizing buffer and cellular apoB100 was immunoprecipitated under the conditions described in the figure legends. The gels were fixed and saturated with Amplify (Amersham Pharmacia Biotech) before being dried and exposed to Kodak Hyperfilm at −80°C for 1-4 days. Films were developed and quantitative analysis of apoB100 bands was performed using an imaging densitometer.23 Following treatment of McA-RH7777 cells with 5 mM GLS or 5 μg/mL TM for 4 hours in the presence or absence of 1 mM PBA, total RNA was extracted Pomalidomide using a commercially available kit (RNeasy; Qiagen). First-strand cDNA was synthesized from 5 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen).24 The resulting cDNA was subjected to 28 cycles of polymerase chain reaction (PCR) amplification (denaturation at 95°C for 30 seconds; annealing at 55°C for 60 seconds; extension at 72°C for 90 seconds). The primer pairs used for detecting messenger RNA (mRNA) levels are listed in Supporting Table 1. Following 24-48 hours transfection, cells were fixed with precooled 100% methanol for

5 minutes and then permeabilized with 0.1% Triton X100 in PBS for 4 minutes. Cells were incubated with rabbit anti-hamster apoB antibody (1:1000) for 1 hour at room temperature or overnight at 4°C in 5% bovine serum albumin. Secondary antibody used in this study was CyTM3-conjugated affiniPure Donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratory, Inc.), dilute 1:500 for 1 hour. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology; sc3598). Images were captured using a Quorum spinning disk microscope (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu back-thinned electron multiplying charge-coupled device camera, spinning disc head, and Volocity 5 software [Improvision]).