Anti ccn2

Treated rat NP cells were immediately placed on ice and washed with cold PBS. To prepare the total cellular proteins, the cells were lysed with lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, complete protease inhibitor cocktail (Roche, IN, USA), 1 mM NaF, and 1 mM Na₃VO₄. Heat-denatured samples were separated on sodium dodecyl sulfate polyacrylamide gels and electrotransferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were then blocked with blocking buffer (5% BSA and 0.1% NaN₃ in PBS) and subsequently incubated overnight at 4 °C with anti-CCN2 (1:1000, Santa Cruz Biotechnology or Abcam, Cambridge, UK) antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Tokyo, Japan). Chemiluminescent signals were visualized with an Immobilon Western Chemiluminescent HRP Substrate (Millipore) and scanned using an Ez-Capture MG imaging system (ATTO, Tokyo, Japan). The western blot data were quantified using Image J pixel analysis (NIH Image software). Western blot data are presented as band intensity normalized to that of the loading control (β-actin). To measure the band intensity, the data shown are representative of at least three independent experiments.