Donkey anti goat hrp conjugated igg

Tumor cells infected by recombinant VVs were lysed in buffer: 50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl containing 0.1% SDS, 1x complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and 1 mM PMSF. Samples (30 μg) were separated by 10% SDS-PAGE and transferred to a Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories) by a wet blotting procedure (100 V, 500 mA, 90 min, 15°C) using “Mighty Small Transphor” (GE healthcare Bio-Science AB, USA). Immunodetection was performed using iBind system (Life Technologies), iBind Cards (Invitrogen, Thermo Fisher Scientific) and antibodies: ANTI-FLAG BioM2 antibody (Sigma-Aldrich, F9291), BAX (1 : 1000, Abcam), cyclin B1 (1 : 60, Sigma-Aldrich), tubulin (1 : 200, Sigma-Aldrich), GAPDH (1 : 100, R&D), and HMGB1 (1 : 6000, Abcam) and using goat anti-mouse HRP-conjugated polyclonal IgG (1 : 200, Abcam) or donkey anti-goat HRP-conjugated IgG (1 : 200, R&D) as secondary antibodies, with Novex ECL HRP chemiluminescent substrate reagent kit (Invitrogen, USA). A C-DiGit blot scanner (Li-COR Bioscience) was used for luminescent detection. Densitometric analysis of the western blot data was performed using the image analysis software Gel-Pro Analyser (Media Cybernetics) version 3.1.

Cisplatin, doxorubicin, everolimus (afinitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phycoerythrin (PE)-conjugated mouse anti-human CD44 monoclonal (#MHCD4404) and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD24 monoclonal (#MHCD4201) antibodies were purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA). FITC-conjugated mouse anti-human HER2 monoclonal (#2222020), FITC-conjugated mouse anti-human CD326 monoclonal (#2221020), PE -conjugated mouse anti-human CD324 monoclonal (#2220530), PE -conjugated mouse anti-human CD325 monoclonal (#2354025), allophycocyanin (APC)-conjugated mouse anti human HER3 monoclonal (#2223540), APC-conjugated mouse anti human CD146 monoclonal (#2310060) antibodies were purchased from Sony Biotechnology Inc. (San Jose, CA, USA). FITC-, APC-, and PE-conjugated IgG controls were from BD Biosciences. Vimentin mouse monoclonal (ab8069) and rabbit polyclonal to Ki-67 (ab15580) antibodies were from Abcam (Cambridge, United Kingdom). Secondary antibodies Donkey Anti-Mouse IgG H&L (ab97029, Abcam), Alexa Fluor^® 594 goat anti-rabbit IgG1 (Thermo Fisher Scientific), goat anti-mouse HRP-conjugated polyclonal IgG (Abcam) and donkey anti-goat HRP-conjugated IgG (R&Dsystems) were used.

Tumor cells were lysed in buffer: 50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl containing 0.1% SDS, 1× complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and 1 mM PMSF. Samples (30 μg) were separated by 10% SDS-PAGE and transferred to a Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories) by a wet blotting procedure (100 V, 500 mA, 90 min, 15 °C) using “Mighty Small Transphor” (GE healthcare Bio-Science AB, USA). Immunodetection was performed using iBind system (Life Technologies), iBind Cards (Invitrogen, Thermo Fisher Scientific) and antibodies: Vimentin mouse monoclonal antibody (1:50), GAPDH goat polyclonal antibodies (1:133), goat anti-mouse HRP-conjugated polyclonal IgG (1:200, Abcam) or donkey anti-goat HRP-conjugated IgG (1:200, R&D systems) as secondary antibodies, with Novex ECL HRP chemiluminescent substrate reagent kit (Invitrogen, USA). A C-DiGit blot scanner (Li-COR Bioscience) was used for luminescent detection. Densitometric analysis of the western blot data was performed using the image analysis software Gel-Pro Analyser (Media Cybernetics) version 3.1.