Phospho p44 42 map kinase

After transfection, cells were washed twice with ice-cold PBS, lysed and separated to cytoplasmic and nuclear fractions using the Nuclear Extract kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA, USA). To detect Akt, phosphorylated Akt, ERK, phosphorylated ERK, NF-κB, phosphorylated NF-κB and MMP-9 proteins we separated these by SDS polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes and transferred proteins to a membrane. Western blot analyses were performed with various specific primary antibodies including Akt (9272), phospho-Akt (Ser473; 9271), NF-κB (3034), phospho-NF-κB (Ser536; 3033), p44/42 MAP kinase (Erk; 4695), phospho-p44/42 MAP kinase (p-Erk; 9101), MMP-9 (2270), β-actin (4970), E-cadherin (3195) (all from Cell Signaling, Danvers, MA, USA) and N-Cadherin (ab18203; Abcam, Cambridge, MA, USA). The immunoreactive bands in the immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit immunoglobulin using an enhanced chemiluminescence western blotting system (ECL Plus; GE Healthcare Life Sciences, Pittsburgh, PA, USA). Non-specific antigen sites were blocked with 10% bovine serum albumin in 1X Tris-buffered saline.