Horseradish peroxidase hrp conjugated anti mouse or anti rabbit secondary antibodies

Protein extracts were resolved on 12% and 10% SDS–PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and blocked in 5% skim milk. The following primary antibodies were used according to the manufacturer's instructions: anti-CD133 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ADAM17 (ab cam), NICD (Cell Signaling Technology, Beverly, MA, USA), MTSS1 (Santa Cruz Biotechnology), HES1 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich Co.). After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, Cardiff, UK), specific protein bands were visualized using enhanced chemiluminescence (Amersham Biosciences). The density of each band was measured using the TINA software.

Western blotting was performed as previously described (93 (link)). hBMECs were starved overnight and collected or stimulated with 100 ng/ml CCL5 for 10 min before harvest. Cells were washed with phosphate-buffered saline (PBS) and lysed in 1% NP-40 buffer with a protease inhibitor cocktail (Sigma) as previously described (12 (link)). Total protein levels were determined in a bicinchoninic acid assay (Thermo Fisher), and proteins were resolved by SDS–12% PAGE, transferred to nitrocellulose, blocked in PBS–1% bovine serum albumin (BSA), and incubated with antibodies in a blocker. Proteins were detected using horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham) and the Luminata Forte HRP substrate (Millipore).

Cells were lysed in lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100 and protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Extracted protein amounts were quantified using the BCA protein assay kit (Pierce). Equivalent amounts of protein (25 μg) were separated using 12% SDS-PAGE and transferred to PVDF membrane (Millipore Corporation, Billerica, MA). For immunoprecipitation, cell lysates were pre-cleared with protein A-Sepharose CL-4B (Amersham Biosciences, Uppsala, Sweden), then incubated overnight at 4°C with Myc or Flag antibody and protein A-Sepharose. The immunoprecipitates were washed three times with lysis buffer and analyzed by Western blot analysis, which was performed using primary antibodies against G6PD (Abcam, Cambridge, UK), BAG3 (Abcam, Cambridge, UK) or GAPDH (Chemicon, Bedford, MA), horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, UK) and ECL solutions (Amersham Biosciences, UK).