Immuno blot nitrocellulose membranes

Protein lysates from MB primary cultures and frozen operative tissue were prepared in lysis buffer (50 mM HEPES, pH7.0, 150 mM NaCl, 2% SDS, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). Proteins were separated on 4–20% Bis-Tris gradient polyacrylamide gels (Invitrogen) and transferred onto Immuno-Blot nitrocellulose membranes (Bio-Rad Laboratories). Membranes were then incubated in blocking buffer (1X TBS containing 5% milk and 0.05% Tween-20, 1 hour), probed overnight with antibodies specific for HNRNPA2/B1 (Cell Signaling, 1:1000), HNRNPA1 (4B10; Santa Cruz; 1:2000), HNRNPC1/C2 (4F4; Santa Cruz; 1:5000), cMYC (9E10; Santa Cruz; 1:500) and actin (Sigma; 1:2000).

To obtain total cell lysates, at the end of the experiment attached cells were scraped off and incubated for 10 min on ice with lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA, 1 mM PMSF and 5 µg/ml leupeptin). After protein content determination with Bradford reagent, total protein was boiled in Laemmli sample buffer and separated by 8% SDS-PAGE. Proteins were then transferred to Immunoblot nitrocellulose membranes (Bio-Rad) and, after blocking, incubated overnight with the indicated primary antibodies: anti-AKT (9272), anti-phospho ERK1/2 (9101), anti-ERK1/2 (9102) and anti-phospho IGF1R (3021) from Cell Signaling Technology (Boston, MA, USA), anti-phospho IRS1 (07-844) from Merk Millipore (Darmstadt, Germany) and anti-phospho AKT (sc-7985-R) and anti-IGF1R (sc-713) from Santa Cruz Biotechnology. Immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad).