Horseradish peroxidase hrp conjugated secondary anti mouse or rabbit antibodies

Whole cell lysates of DU 145 were prepared by using lysis buffer (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 mM iodoacetamide, 2 mM PMSF). To measure the protein contents, a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA) was used. The proteins were separated on 10% tris-glycin gels, and electrotransferred onto a Hybond ECL transfer membrane with transfer buffer (25 mM Tris, 250 mM glycine, 20% methanol). The membranes were blocked in 5% nonfat dry milk in TBS buffer containing 0.1% Tween 20 (TBST) and immunoblotted with antibodies of cleaved caspase 3, cleaved caspase 8, procaspase 8, procaspase 9, PARP, Bcl-2, Bcl-_XL, Bax, DR 5, FLIP and FADD (Cell signaling, Beverly, MA, USA), and β-actin (Sigma Aldrich Co., St. Louis, MO, USA), and then exposed to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (AbD Serotec, Raleigh, NC, USA). Protein expression was examined by using enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ, USA).

PC-3 or DU145 cells treated by TRAIL and/or Tanshinone I were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid, 1 M EDTA, 1 mM Na₃ VO₄, 1mM NaF and protease inhibitors cocktail). To quantify protein samples, Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA) was used. Proteins samples were separated by electrophoresis on SDS-PAGE gel and electrotransferred onto a Hybond ECL transfer membrane (Amersham Pharmacia, Piscataway, NJ, USA). After blocking, the membrane was incubated with primary antibodies for PARP,caspase8, caspase 9, AKT, p-AKT, ERK, pERK, cleaved caspase 3, Bcl-X_L, Bcl 2, Bax, DR5 and beta actin (Cell signaling, Beverly, MA,USA) followed by exposing to horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (AbD serotec, kidlington, UK). To visualize protein bands, chemiluminescence (ECL) system (Amersham Pharmacia, Piscataway, NJ, USA) was used.

The brain tissues was added with PBS buffer (300 μL), homogenized with homogenizer and centrifuged 800 rpm, 10 s. The supernatant was lysed with lysis buffer 50 μL for 30 min in the ice and centrifuged at 14,000 rpm at 4 °C for 20 min. The protein concentrations were calculated using the Bradford assay [50 (link)]. The same amount of proteins were separated by electrophoresis on SDS-PAGE gel and electrotransferred onto a NC (nitro cellulose) membrane using a semi-dry transfer system (Bio-Rad, Hercules, CA, USA). After blocking with blocking buffer (0.5% skim milk, 1× PBST buffer) for 1 h and washing with 1× PBST buffer for 10 min (three times), the membrane was exposed to primary antibodies for BDNF (Santa Cruz, CA, USA), p-CREB (Ser133), total-CREB (Santa Cruz, CA, USA) and GAPDH (Cell signaling, Beverly, MA, USA). After 2 h, membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse or rabbit antibodies (AbD Serotec, Kidlington, UK). The enhanced chemiluminescence (ECL) system (Amersham Pharmacia, Piscateway, NJ, USA) of Western Blot detection kit (Bio-Rad, Hercules, CA, USA) was used to visualize protein bands with Chemi-Doc (Bio-Rad, Hercules, CA, USA).