Rabbit polyclonal anti erk1 2

Total protein was prepared from cells or dispase-separated epidermis and then quantified by the Bradford method. Briefly, the detergent-soluble fractions were subjected to SDS-PAGE according to a standard protocol. Separated protein bands were transferred to a nitrocellulose filter. Membranes were blocked with 5% skim milk powder at room temperature for 2 h and incubated overnight with primary antibody at 4°C. Detection of the secondary antibody was performed using the ECL Plus kit (Amersham Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. Primary antibodies used in Western blotting included mice monoclonal anti-TNIP1 (1:1000 dilution, USA), rabbit polyclonal anti-cytokerotin (CK) 6 (1:1000 dilution, GeneTex), rabbit polyclonal anti-C/EBPβ (1:1000 dilution, GeneTex), rabbit polyclonal anti-Erk1/2(1:1000 dilution, GeneTex), rabbit monoclonal anti-p-Erk1/2 (1:1000 dilution, Cell Signaling Technology, USA), and rabbit polyclonal anti-TNIP1 (1:1000 dilution, LSBio, USA) antibodies. HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The images were captured and analyzed using Quantity One software.

Proteins were run in 10% SDS‐polyacrylamide gels and transferred to supported nitrocellulose membranes by standard techniques. Membranes were incubated with the mouse monoclonal anti‐VEGFR‐1 (clone D2, 1:500; Santa Cruz Biotechnology), rabbit polyclonal anti‐Erk1&2 (1:1000; Genetex), rabbit polyclonal anti‐phospho‐Erk1&2 (Thr/Tyr185/187, 1:1000; Invitrogen) or rabbit polyclonal anti‐β‐tubulin (1:10 000; Santa Cruz Biotechnology) as primary antibodies. Immunodetection was performed using antimouse or anti‐rabbit Ig/Horseradish peroxidase secondary antibodies and ECL Western blotting detection reagents from GE Healthcare (Milan, Italy).