Rabbit monoclonal anti p erk1 2

In order to further investigate the possible mechanisms of NSCs’ effect on tumor cell behavior changes, expression of mutant p53, caspase-3 and the phosphorylation status of ERK1/2, AKT were detected by Western blot assay. Rabbit monoclonal anti-mutant p53 (1:1,000, abcam, USA), rabbit monoclonal anti-GFAP (1:2,000, Millopore, USA), rabbit polyclonal anti-caspase 3 (1:1,000, Cell Signaling, USA), rabbit polyclonal anti-AKT (1:1,000, Cell Signaling, USA), rabbit monoclonal anti-p-AKT (1:1,000, Cell Signaling, USA), rabbit monoclonal anti-ERK1/2 (1: 1,000, Cell Signaling USA), rabbit monoclonal anti-p-ERK 1/2 (1: 1,000, Cell Signaling USA) were used.

Western blotting analysis was performed as previously described [62 (link)] using the following primary antibodies: rabbit polyclonal anti-TH (ab112, 1:200), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; ab7260, 1:1500), rabbit polyclonal anti-c-FOS (ab7963, 1:500), rabbit polyclonal anti-IL-1β (ab9722, 1:500), mouse monoclonal anti-IL6 (ab9324, 1:500), and rabbit polyclonal anti-TNF-α (ab6671, 1:500) (all from Abcam, Cambridge, MA, USA); rabbit monoclonal anti-ERK1/2 (#4695, 1:1000), rabbit monoclonal anti-p-ERK1/2 (#4377, 1:500) (both from Cell Signaling Technology, Danvers, MA, USA); and rabbit polyclonal anti-Gsto2 (14562-1-AP, 1:1000) and rabbit polyclonal anti-PTGER3 (14357-1-AP, 1:500) (both from Proteintech, Rosemont, IL, USA). Rabbit monoclonal anti-GAPDH antibody (Abcam; ab181602, 1:3000) was used for the loading control. Protein band density was quantified using an Epson V330 Photo scanner (Seiko Epson, Nagano, Japan) and Quantity One software (Bio-Rad, Hercules, CA, USA).

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.