Protein Extraction and Immunoprecipitation Protocol

Cell protein extracts were prepared using modified RIPA buffer (50 mM Tris HCL pH 7.4, 0.5–1.0 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl with freshly added complete protease (Roche, Mannheim, Germany)), as described previously [13 (link)]. Phosphatase inhibitor (Active Motif, Rixensart, Belgium) and MG-132, a proteasome inhibitor, were used as needed. Nuclear extraction was performed using the Nuclear Extraction Active Motif kit (Active Motif). Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). Immunoprecipitation was conducted with anti-PTP-RR Ab (Abcam) or anti-PP2A Ab (Bethyl, Montgomery, TX). Protein extracts or immunoprecipitates were separated by SDS-PAGE (Invitrogen, Paisley, UK) and detected by Western Blot analysis using chemiluminescence (ECL Plus; GE Healthcare, Chalfont St. Giles, UK). β-actin expression was used as a control for protein loading as needed.