10494 1 ap

Western blotting was performed as previously described^{49 (link)}. Briefly, cells were lysed with RIPA buffer containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF) to obtain the protein. Equivalent amounts of denatured proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 10% gel and transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with a primary antibody followed by a second antibody for the chemiluminescence detection. The specific antibodies in this approach were: the LMP1 monoclonal antibody (mAb) (DAKO Lifetech, Glostrup, Denmark, M0897), anti-p-NF-κB (p65) antibody (CST, Chicago, USA, #3033), anti-NF-κB antibody (rabbit mAb, Millipore, 06-418), anti-CDH6 antibody (mouse mAb, Millipore, MAB2013), anti-RUNX2 rabbit mAb (Abcam, Cambridge, UK, AB23981), the antibodies of EMT makers (anti-E-cadherin, anti-Vimentin, anti-N-cadherin, anti-Slug and anti-Snail antibody, CST, Chicago, USA, #9782), anti-GAPDH antibodies (Proteintech, Chicago, USA, 10494-I-AP) and β-actin antibodies (Proteintech, 66009-I-Ig).

The membrane-permeable 8-Br-cGMP (8-bromoguanosine 3′,5′-cyclic monophosphate, B1381) and 8-Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate, B5386) were from Sigma. ANP (AS-20648), BNP (AS-24016), and CNP (AS-24244) were from AnaSpec. Antibodies used in this study included anti-p-Ser239 VASP (3114, Cell Signaling Technology), anti-total VASP (3112, Cell Signaling Technology), anti-GAPDH (sc25788, Santa Cruz; 10494-I-AP, ProteinTech), anti-rabbit IgG conjugated with alkaline phosphatase (A3687, Sigma).