Anti p27 antibody

Biotinylated-BSA (1 μg/μl, Thermo) is flowed for 25 min through the device, allowing its binding to the epoxy surface. On top of the biotinylated-BSA, 0.5 μg/μl of NutraAvidin (Pierce, Rockford, IL) is added (flow for 20 min). The ‘button’ valve is then closed, and biotinylated-PEG (1 μg/μl, (PG2-AMBN-5k, Nanocs Inc.) is flowed over for 20 min, passivating the flow layer, except for the buttons area. Following passivation, the ‘button’ valve is released and a flow of 0.2 μg/μl biotinylated anti-GFP antibodies (Abcam; #ab6658, Cambridge, United Kingdom) or 0.01 µg/µl biotinylated anti-Flag antibodies (Cell Signaling; #2908 S Danvers, MA, USA) were applied. The antibodies bound to the exposed NutraAvidin, specifically to the area under the ‘button’, creating an array of anti-GFP - or anti-Flag tag. PBS buffer was used for washing in between steps. In the case of p27 immobilization, surface chemistry was performed with 0.2 μg/ml donkey anti-mouse whole biotinylated anti-IgG antibodies (#715-065-150, Jackson Immuno research laboratories, Maryland, USA) followed by 20 min flow of 6.5 μg/ml anti p27 antibodies (Santa Cruz biotechnology, Heidelberg Germany; #1641 mouse).

Antibodies against p‐STAT3 (pY705), p‐AKT (pS473), p‐ERK1/2 (Thr202/Tyr204), p‐MEK1/2 (pS221/221), p‐BRAF (pS445), AKT, ERK1/2, MEK1/2, cyclin D, cyclin E, p‐S6 (pS240,244), Bcl‐2, Bcl‐XL, Bim, and active caspase 3 were purchased from Cell Signaling Technology (Beverley, MA, USA). An anti‐p27 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti‐MIF antibody was purchased from R&D Systems (Minneapolis, MN, USA). Subconfluent cells (70–80%) were used for protein analyses. The cells were treated under various conditions as described. Cells were lysed in RIPA buffer on ice for 15 min (50 mmol·L⁻¹ Tris/HCl, pH 7.5, 1% NP‐40, 0.1% Na deoxycholate, 150 mmol·L⁻¹ NaCl, 0.1 mmol·L⁻¹ aprotinin, 0.1 mmol·L⁻¹ leupeptin, 0.1 mmol·L⁻¹ pepstatin A, 50 mmol·L⁻¹ NaF, 1 mmol·L⁻¹ sodium pyrophosphate, 1 mmol·L⁻¹ sodium vanadate, 1 mmol·L⁻¹ nitrophenolphosphate, 1 mmol·L⁻¹ benzamidine, and 0.1 mmol·L⁻¹ PMSF) and centrifuged at 12 000 g for 20 min. Samples containing equal amounts of total protein were resolved in SDS polyacrylamide denaturing gels, transferred to nitrocellulose membranes, and probed with antibodies. Detection was performed using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK).