Rabbit polyclonal anti p21

Cell lysis and Western blot analysis were performed as described previously [40 (link)], using the following antibodies: Rabbit polyclonal anti-SIK2 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-SIK2-pS385 (Kinexus, Sydney, Australia), rabbit polyclonal anti-AKT (Cell Signaling), rabbit polyclonal anti-AKT-pS473 (Cell Signaling), mouse monoclonal anti-β-actin (Sigma-Aldrich), rabbit polyclonal-anti KIF18B (ThermoFisher scientific, Waltham, MA, USA), rabbit polyclonal anti-EB1 (Abcam, Cambridge, UK), mouse monoclonal anti-cyclin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-Histone 3-pS10 (ThermoFisher scientific), mouse monoclonal anti-PLK1 (Santa Cruz Biotechnology), mouse monoclonal anti-vinculin (Santa Cruz Biotech), mouse monoclonal anti-P53 (Santa Cruz Biotech), rabbit polyclonal anti-P21 (cell signaling), rabbit polyclonal anti-BCL-xL (Santa Cruz Biotechnology), rabbit polyclonal anti-Cyclin D (Santa Cruz Biotechnology).
Reagents were purchased from the following sources: Paclitaxel (T7402) Sigma-Aldrich, propidium iodide (440300250) Acros Organics (Fair Lawn, NJ, USA), RNase A (1007885) Qiagen (Hilden, Germany), PE Annexin V (556421) and 7AAD (21-68981E) BD Biosciences, Caspase-Glo^® 3/7 Assay system (P1781) Promega, RO3306 (S7747) Selleckchem (Houston, TX, USA).

MDA-MB-231 cells were seeded in 6-well plates at a density of 1.5 × 10⁵ cells/well for 24 h, followed by treatment with 16 µM of compound 7. Protein extracts were quantified using the Bradford reagent (Sigma-Aldrich). Proteins were run in SDS-PAGE and transferred to a Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). After blocking, proteins were identified using specific primary antibodies: rabbit polyclonal anti-p21 (Santa Cruz Biotechnology, Dallas, DX, USA), rabbit polyclonal anti-Bax, and rabbit monoclonal anti-Bcl-xL (Invitrogen, Waltham, MA, USA), followed by the respective anti-mouse/anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). GAPDH was used as a loading control. The signal was detected with the ECL Amersham kit from GE Healthcare (VWR, Carnaxide, Portugal). Two detection methods were used: the Kodak GBX developer and fixer (Sigma-Aldrich) or the ChemiDoc™ XRS Imaging System from Bio-Rad Laboratories (Amadora, Portugal).

Cells were lysed in buffer consisting of 50 mM Tris-HCl pH 8, with 1% NP-40 (Igepal AC-630) 150 mM NaCl, 5 mM EDTA and fresh protease inhibitors. Protein concentrations were determined by colorimetric assay (Bio-Rad). Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Tubulin (Santa Cruz Biotechnology), mouse monoclonal anti-Gapdh (Santa Cruz Biotechnology), rabbit polyclonal anti-p21 (Santa Cruz Biotechnology), rabbit polyclonal anti-Cyclin A (Santa Cruz Biotechnology), mouse monoclonal anti-Cyclin E (Santa Cruz Biotechnology), rabbit monoclonal anti-EGFR (Cell Signaling Tecnology, C74B9), rabbit polyclonal anti-N-Cadherin (Abcam). Secondary antibodies used were goat anti-mouse and goat anti-rabbit conjugated to horseradish peroxidase (Santa Cruz Biotechnology).