Ji-Young Kim, et al. Table 1. Primer Sequences and RT-PCR Conditions Gene of interest Primer PCR conditions Bax (S) (AS) Bcl-2 (S) (AS) Bcl-XL (S) (AS) β-actin (S) (AS) 5'-AAGCTGAGCGAGTGTCTCCGGCG-3' 5'-GCCACAAAGATGGTCTGCC-3' 95 (10 min) - 95 (30 s) - 64 (30 s) -72 (30 s) - 72 (7 min) 5'-CAGCTGCACCTGACGCCCTT-3' 5'-GCCTCCGTTATCCTGGATCC-3' 95 (10 min) - 95 (1 min) - 52 (1min) -72 (1 min) - 72 (7 min) 5'-TGGAGTAAACTGGGGGTCGCATCG-3' 95 (10 min) - 95 (1 min) - 50 (1 min) - 72 (1 min) 5'-AGCCACCGTCATTCCCGTCAGG-3' - 72 (7 min) 5'-CCAGAGCAAGAGAGGTATCC-3' 5'-CTGTGGTGGTGAAGCTGTAG-3' 95 (10 min) - 95 (45 s) - 58 (2 min 30 s) - 72 (10 min) 7.5, 60 mM beta-glycerophosphate, 10 mM EDTA, 10 mM MgCl2, 10 mM NaF, 2 mM DTT, 1 mM Na3VO4, 1 mM APMSF, 1% Np-40, 5 μg/mL leupeptin). Following a 40 min incubation on ice, the lysates were combined with 2 X sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.2% bromphenol blue) and boiled for 5 min, samples were resolved on a SDS-polyacrylamide gel. After semi-dry transfer to a PVDF membrane (Amersham Pharmacia Biotech, Uppsala, Sweden), ponceau S staining was used to confirm loading of equal amounts of total protein loaded in each lane. The membranes were blocked (5% nonfat dried milk, 0.1% Tween 20, 100 mM NaCl, 10 mM Tris-HCl, PH 7.6) for 1 h and incubated with the appropriate primary antibody: monoclonal mouse anti-Bax antibody (BD PharMingen, San Diego, CA, USA, 2 μg/mL), polyclonal rabbit anti-Bak antibody (BD Phar Mingen, 2 μg/mL), monoclonal mouse anti-Bcl-2 antibody (Oncogene, San Diego, CA, USA, 2 μg/ mL), polyclonal rabbit anti-caspase 3 antibody (Cell Signaling, Beverly, MA, USA, 1:100), mono- clonal mouse anti-poly (ADP-ribose) polymerase (PARP) antibody (BD Phar Mingen, 1:200), polyclonal rabbit anti-caspase 8 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200), or polyclonal rabbit anti-actin antibody (Santa Cruz Biotechnology, 1:200). Bound primary anti- bodies were visualized with an ECL anti-mouse IgG peroxidase-linked species-specific F(ab')2 frag- ment (Amersham Pharmacia Biotech, 1:10,000) or an ECL anti-rabbit IgG peroxidase-linked species- specific F(ab')2 fragment (1:200,000) and the ECL PLUS chemiluminescence detection system em- Yonsei Med J Vol. 47, No. 6, 2006 ployed according to the manufacture's instruc- tions (Amersham Pharmacia Biotech). Western blot for NF-κB and IκBα Nuclear and cytoplasmic fractions were pre- pared from spleen cells.10 Monoclonal mouse IκBα antibody (Cell signaling, 1:100), rabbit phospho- NF-κB p65 antibody (Cell signaling, 1:50), NF-κB p65 antibody (Cell signaling, 1:100) and mouse anti-actin antibody (Chemicon international, Temecula, CA, USA, 1:1,000) were used for detec- tion of NF-κB and IκBα by Western blot as de- scribed above.11 Electrophoresis motility shift assay (EMSA) Nuclear extracts from spleen cells were pre- pared as previously described.10 Protein concen- trations of the nuclear extracts were determined by Bradford assay, and EMSAs were performed according to the manufacture's instructions (Promega, Madison, WI, USA). Briefly, 5 μg of nuclear extract was incubated with a (γ32P)-labeled oligonucleotide probe corresponding to the con- sensus NF-κB binding site for 30 min at room temperature. After incubation, bound and free DNAs were resolved on a 5% polyacrylamide gel12 run at 100 V for 3 h, dried, and subjected to autoradiography for 12 h to 48 h.13 Transmission electron microscopy (TEM) The apoptotic changes of mouse spleen cells treated with AD (10 μg/mL) and/or infected with