Westernbright sirius kit

The enriched fraction of the intestinal brush border was prepared following the procedure previously described [23 (link)]. The brush border-enriched fraction and the cell lysate (60 μg of total protein) from Caco-2/TC7 cells were electrophoresed in 8 % SDS-PAGE gels and then transferred to PVDF membranes by electroblotting. The membranes were blocked with 5 % nonfat dried milk plus 1 % BSA and probed using a specific primary antibody (anti-SERT 1:500, anti-TLR2 1:1000, and anti-TLR10 1:1000). The primary antibody was detected using a secondary antibody coupled to horseradish peroxidase and the WesternBright Sirius Kit (Advansta, Menio Park, CA) and was visualized with VersaDoc (Imaging System, Bio-Rad). The blots were reprobed after stripping, with goat polyclonal anti-human β-actin used to determine differences in the sample load. The specific protein/β-actin protein ratio was calculated in densitometric units from the film with Quantity One Analysis Software (Bio-Rad), and the results were expressed as a percentage of the control values.

To confirm drug resistance state, cells were seeded and then exposed to different concentrations of trastuzumab. After double wash with cold PBS, the monolayers were scraped into lysis buffer (Complete Lysis-M kit, Roche) and protein isolation was completed according to the instructions manual. The protein concentration was determined using Coomasie Plus Bradford Assay (ThermoScientific) and used to estimate the volume of protein yielding 15 μg, which were boiled in Laemmli buffer and resolved on an 8% SDS-PAGE and transferred onto a PVDF membrane (L-08008-001, Advansta). Primary antibodies were: anti-ErbB2/HER2 (1:1000, ab8054, Abcam) and anti-Beta-actin (1:1000, 634801, Biolegend) was served as a loading control. Mouse secondary antibody (1:10,000, 7076S; Cell Signaling) conjugated with HRP (horseradish peroxidase) was added and kept at room temperature for 60 min. The immunoblot was developed with the WesternBright Sirius Kit (K-12043-D20, Advansta).

The cells were lysed using M-PER protein lysis buffer (Thermo Fisher Scientific, Inc.). Protein determination was performed using the bicinchoninic method [38 (link)], and the 40 µg proteins of the total cell lysates were run on SDS-polyacrylamide gels followed by western blotting using standard procedures. A WesternBright Sirius Kit (Advansta, CA, USA) was used for antibody detection, and an AE-9300 Ez capture MG (ATTO, Tokyo, Japan) was used for image data capture. We repeated the western blot analysis three times and the results were similar.

To monitor the production of the scFv BL1 and hGH, proteins in whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by either Coomassie blue staining or immuno-blotting. scFv BL1 was analyzed on Tris Glycine 8–16% (Invitrogen) gels and hGH was analyzed using Tricine 16% (Invitrogen) gels. Lysates equivalent to 0.04 A₆₀₀ units were loaded onto gels for Coomassie staining and lysates equivalent to 0.02 A₆₀₀ units were loaded onto gels that were analyzed by immuno-blotting. For volumetric comparisons, cell pellets derived from 1 ml of culture were solubilized in 300 μl of sample buffer and 8 μl was loaded for Coomassie blue staining and 4 μl for immuno-blotting. In the blotting experiments, secretory/secreted BL1 and hGH were detected based on their C-terminal His₆-tag using HisProbe-HRP Conjugate (Pierce) or the AlexaFluor 647 conjugated anti-HIS antibody (Invitrogen). The HisProbe-HRP Conjugate was used for mere detection and the AlexaFluor 647 conjugated anti-HIS antibody for quantification. The Western Bright Sirius kit (Advansta) was used to visualize immuno-blots stained with the HisProbe-HRP Conjugate. Chemiluminescence and fluorescence signals were detected using an Azure c600 imaging system (Azure Biosystems).

Cells in culture were lysed using Complete Lysis-M kit (Roche). The protein concentrations of the lysates were quantified using Coomassie Plus Protein Assay Reagent (Thermo Scientific). 10 ug of protein for each sample were loaded on to 8% SDS-PAGE gel. Separated proteins were transferred to a PVDF membrane (L-08008-001, Advansta) in wet transfer buffer. Membranes were blocked with 5% milk in TBST (0.5%) for 1 hour at room temperature before incubation at +4°C overnight with the following antibodies: HER2 (1:1000, ab8054, Abcam), total AKT (1:1000, sc8312, SantaCruz), phospho-AKT (Ser 473, 1:1000, sc-7985-R, SantaCruz), total ERK2 (1:1000, sc-154, SantaCruz), phosho-ERK 1/2 (Thr 202 / Tyr 204, 1:1000, sc-81492, SantaCruz) and beta-actin (1:1000, 634801, Biolegend) in 3% milk powder-TBST. After incubation with HRP-conjugated secondary antibodies, the protein bands were detected using WesternBright Sirius Kit (K-12043-D20, Advansta).