Goat anti rabbit and horseradish peroxidase conjugated igg

In this study, sinularin was obtained from National Museum of Marine Biology & Aquarium (Pintung, Taiwan) and the chemical structure showed in Figure 1B. The anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA), apoptosis antibody sample kit, pro-apoptosis bcl-2 family antibody sample kit, pro-survival Bcl-2 family antibody sample kit, phospho-pI3 kinase p85 (Tyr458)/P55 (Tyr799) antibody, phosopho-GSK3β (Ser9) (D3A4) rabbit antibody, PI3 kinase p110α antibody were obtained from Cell Signaling (Danvers, MA, USA), anti-cytochrome C, CD95/Fas, AIF, phospho-Akt1, Akt1, mTOR/FRAP and phosopho-mTOR (Phospho-Ser2481) antibody were obtained from Epitomics (Bellerica, MA, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained for Millipore (Bellerica, MA, USA).

Rabbit anti-human Bax, ERK, JNK and p-JNK antibodies were obtained from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against p-PI3K, AKT, p-AKT, PARP, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, Bcl-xl, Bcl-2, Mcl-1, Bad, p38, p-p38, p-ERK, p-ATF2 and p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human antibodies against p-Bad and cytochrome C were obtained from Epitomics (Burlingame, CA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained from Millipore (Bellerica, MA, USA). Immunoblotting was performed as previously described [19 (link)]. The treated samples and the control samples (25 μg) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were then transferred to PVDF membranes. The PVDF membranes were incubated with the primary antibody (1:1000 dilutions in 2% dehydrated skim milk) at 4 °C overnight. Then, incubation at 4 °C was performed for 2 h with the secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk). The blots were detected through chemiluminescence using enhanced ECL western blotting kit.

The stems of Piper betle were collected in Pingtung County, Taiwan in July 2008, which were cultivated by local farmer and bornyl cis-4-hydroxycinnamate identified by Chi-I Chang, National Pingtung University of Science and Technology. Rabbit anti-human MMP-2, MMP-9, uPA, TIMP-1, TIMP-2, FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK were obtained from Cell Signaling Technology (Danvers, MA, USA). GRB2, Rac, PKC, Ras, RhoA, MEKK3, MEKK7, N-cadherin, E-cadherin, Snail, and Lamin A2 antibodies were obtained from Epitomics (Burlingame, CA, USA). Dimethyl sulfoxide (DMSO), protease inhibitor cocktail, and rabbit anti-human β-actin antibodies were purchased from Sigma (St. Louis, MO, USA). PVDF (polyvinylidene difluoride) membranes and goat anti-rabbit and horseradish peroxidase-conjugated IgG were purchased from Millipore (Bellerica, MA, USA). Chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Pierce (Rockford, IL, USA).