Anti hnrnpd

Cytoplasmic lysates were prepared from NIH-3T3 cells using hypotonic-lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 0.2 mM PMSF, and 2.5% NP-40 at 4°C. Nuclei were obtained by centrifugation, and washed twice in the hypotonic-lysis buffer to avoid contamination of cytoplasmic proteins. Nuclear lysates were prepared using extraction buffer containing 20 mM HEPES (pH 7.9), 450 mM NaCl, 1.5 mM MgCl 2 , 1 mM DTT, 0.2 mM PMSF, and 0.2 mM EDTA (pH 8.0) at 4°C. Both lysates were followed by repeated freeze-thaws for complete disruption. Immunoblot analysis was performed using polyclonal anti-PTB, polyclonal anti-PER3, anti-hnRNP D (Millipore Corporation, Bedford, MA, USA), anti-14-3-3f, anti-Lamin-B (Santa Cruz Biotechnology, Dallas, TX, USA), anti-hnRNP K (Abcam, Cambridge, UK) and monoclonal anti-FLAG (Sigma, St Louis, MO, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore Corporation) primary antibodies and horseradish peroxidase-conjugated rat-, rabbit-specific (Bethyl Laboratories, Montgomery, TX, USA), and mouse-specific (Thermo Scientific, Rockford, IL, USA) secondary antibodies. Proteins were analyzed using SUPEX enhanced chemiluminescence (ECL) solution kit (Neuronex, Daegu, Republic of Korea) and LAS-4000 chemiluminescence detection system (Fujifilm, Tokyo, Japan), according to the manufacturer's instructions.