Horse radish peroxidase conjugated goat anti mouse and anti rabbit secondary antibodies

SOX2 (D6D9), OCT4 (C30A3), γH2AX (Ser139), CDK1, CDK2, p15INK4B, Cyclin D1 (DSS6), p-Aurora A (T288), p-Aurora B (T232, 1:1000), Aurora B, c-Myc (9E10) and Phospho-Chk2 (Thr68), Phospho-ATM (Ser198), Phospho p53 (Ser 15) antibodies were purchased from Cell Signalling Technology. GFP (clone B-2) antibody was purchased from Santa Cruz Biotechnology. Primary antibodies are used in 1:200 dilution for Immunofluorescence assay and 1:1000 dilution for Western blotting. GAPDH (clone MAB374, 1:4000) antibody was purchased from Millipore.
Horse radish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000) were purchased from Jackson ImmunoResearch. Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 568 goat anti-rabbit antibody (1:200) were purchased from Life Technologies. Anti-PRL3 monoclonal antibody (mAb) (clone 318, 1:200 for Immunofluorescence and 1:2000 for western blot) was generated in-house. PRL3-zumab was engineered based on the original framework of murine anti-PRL3 mAb (clone 318).

Cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors (Roche). Equal amounts of cell lysates from the protein samples were resolved by 10 and 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. These membranes were incubated with 5% skimmed milk or 5% BSA in PBST at room temperature for 1 h; after rinsing 3 times, the membranes were incubated overnight at 4 °C with the following specific primary antibodies: mouse anti-human CD147 (at 1:1000, Abcam), rabbit anti-human MMP9, rabbit anti-human p38 MAPK and rabbit anti-human phosphorylated p38 MAPK (at 1:1000, Cell Signaling Technology Company). This step was then followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000, Jackson) for 1 h at room temperature. The blots were visualized with an enhanced chemiluminescence kit (ECL plus). The intensity of each band was measured by using Quantity One software. The relative protein expression levels were determined by normalization to that of β-actin.