Bax sc 7480

After treatment of AgNPs for 24 h, total cellular proteins were prepared using RIPA lysis buffer (20–188 Merck Millipore, Germany) (included protease inhibitor cocktail) from MCF7 cells [30 (link)]. The protein concentrations were established by bicinchoninic acid assay (71285-Merck Millipore, Germany). Equal amount of protein was separated by 12% polyacrylamide gels and then transferred onto PVDF membranes (sc-3723, Santa Cruz, USA). The membranes were blocked with 2.5% BSA at 4°C overnight and then incubated with specific primary antibodies (Bax (sc-7480) and Bcl-2 (sc-7382), Santa Cruz, USA). After washing with TBST (containing 0.1% Tween 20) 3 times, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies in TBST at 37°C for 1 h. The protein β-actin (sc-47778) was used as a housekeeping control for normalization. Finally, the expression levels of proteins were visualized and analyzed using ImageJ software.

Cells and tissues were collected, washed twice with PBS, lysed on ice for 30 min in 100 μl lysis buffer and then centrifuged at 13 000g for 15 min. The supernatants were collected from the lysates and the protein concentration was determined. Aliquots of the lysates (15 μg of protein) were boiled for 5 min and electrophoresed using a SDS/10% PAGE. The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad), which were then incubated with primary antibodies. Bax (sc-7480, 1:200), Bcl-xL (sc-8392, 1:200), Bcl-2 (sc-783, 1:200), phospho-Bcl-2 (Ser⁸⁷) (sc-16323, 1:200) and β-actin (sc-103656, 1:1000) antibodies were purchased from Santa Cruz Biotechnology. The nitrocellulose membranes were further incubated with secondary immunoglobulin-G-HRP conjugates. Immunostaining was detected using an ECL® (enhanced chemiluminescence) system (Amersham Biosciences). Densitometric quantification of the target proteins was performed with a β-actin control using Scion Image (Version 4.0.3.2, Scion Corporation) for Windows.

HSC-3 and OECM-1 cells were treated with DMSO or test agents for 24 h. Whole-cell lysates were washed twice by PBS and lysed in RIPA buffer, containing 1% protease inhibitor cocktail and phosphatase inhibitor. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After blocking with nonfat milk for 1h at room temperature, membranes were hybridized with primary antibodies at 4 °C overnight. The blot was then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein expression signals were detected using the Multi-function Gel Image System, MultiGel-21(TOPBIO). Bands of β-actin served as a loading control. The following antibodies p21(#2947), PCNA (#13110), CDC2 (#9116), cyclin B1 (#12231), cyclin A2 (#4656), PARP (#9542), Bcl-2 (#15071), cleaved Caspase-3 (#9664) and survivin (#2808) were purchased from Cell Signaling Technology (Beverly, MA, USA). BAX (SC-7480) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and β-actin was bought from BioLegend (San Diego, CA, USA).

BMMC samples exposed to either SMG or 1G for 48 h were resuspended at 5×10⁶ cells/ml, stimulated with P+I for 10 min, washed twice with ice-cold phosphate-buffered saline (PBS), and lysed with radio-immunoprecipitation assay buffer (RIPA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The lysates were quantified using a Bradford protein assay kit (Bio-Rad) and protein samples were separated by SDS-PAGE, transferred onto Nitrocellulose membrane (Bio-Rad), and probed with the following primary antibodies: Bcl-2 (sc-7382) and Bax (sc-7480) from Santa Cruz Biotechnology, and Bcl-xL (#2764), Bax (#7480), BAK (#12105), Vinculin (#13901), phospho (p)-Erk1/2 (#4370), Erk1/2 (#4695), p-Jnk (#4668), Jnk (#9252), p-p38 (#4511) and p38 (#8690) from Cell Signaling Technology. All these antibodies were used at a 1:1000 dilution. Band densities in immunoblots were quantified by densitometry analysis using Adobe Photoshop CS6 (Adobe Systems Software).

RIPA lysis and extraction buffer (Thermo Fisher Scientific, USA) was used for digesting the sugiol treated or untreated U87 cancer cells to isolate the total proteins. After quantification using Bradford assay, 40 μg of total proteins from each sample was resolved for band separation on SDS-PAGE gels. The separated proteins were transferred electrophoretically to nitrocellulose membranes. The membranes were then incubated with primary antibodies (Bax (sc-7480, Santa Cruz, CA, USA), Bcl-2 (sc-23960, Santa Cruz, CA, USA), and actin (sc-58673, Santa Cruz, CA, USA)) at 4°C overnight (dilution for all antibodies were kept 1 : 1000). The proteins were then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (sc-2357-CM; Santa Cruz, CA, USA). The enhanced chemiluminescent substrate was finally used for protein band detection. Human actin protein served as an internal control in protein loading.