Anti phospho met

On a 10% SDS–PAGE gel, 20 μg total protein was electrophoresed, transferred onto to a polyvinylidene fluoride membranes, blocked, incubated with primary antibody (anti-SOCS1, Cell Signaling Technology, Boston, MA, USA; anti-SOCS3, Abcam Biotechnological Co., Cambridge, UK; anti-Met, Proteintech, Rosemont, IL, USA; anti-phospho-Met, Cell Signaling Technology; anti-AKT, Proteintech; anti-phospho-AKT, Cell Signaling Technology; anti-β-actin, Abcam Biotechnological Co; anti-PCNA, Santa Cruz.) and then with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized using a chemiluminescence solution. β-actin was employed as an endogenous control.

Western-Blotting was performed as previously described [37 (link)]. Primary Antibodies included Anti-GLI1 (Rabbit monoclonal, Abcam), Anti-HHIP (Rabbit polyclonal, Genetex), Anti-β-tubulin (Mouse monoclonal, Enogene), Anti-Met (Rabbit monoclonal, Cell Signaling Technology), and Anti-phospho-Met (Rabbit monoclonal, Cell Signaling Technology). Secondary antibodies included Anti-rabbit IgG (Goat polyclonal, Peroxidase conjugated, Merck Millipore) and Anti-mouse IgG (Goat polyclonal, Peroxidase conjugated, Merck Millipore).

Mice xenograft tumors originating from NIH3T3 cell lines that expressed FOXP2 or FOXP2-CPED1 were fixed in 4% paraformaldehyde. Consecutive paraffin sections of the mice xenograft tumors and prostates of ARR2PB-FOXP2 or ARR2PB-FOXP2-CPED1 transgenic mice (4 μm thickness) were used for H&E staining and immunohistochemical analyses. Specimens from 25 cases of benign prostatic hyperplasia and 45 cases of primary prostate cancer were analyzed for FOXP2 staining with an immunohistochemical assay. Protein expression was evaluated as negative, low, medium, and strong. The sections were pretreated with citrate buffer (pH 9.0) in a microwave oven for 20 min for antigen retrieval. The primary antibody to FOXP2 (Sigma, #HPA000382) was used at a 1:200 dilution. The following primary antibodies were used for immunohistochemical staining: anti-Ki67 (Abcam, #ab16667, 1:300), anti-Phospho-Akt (Ser473) (Cell Signaing Technology, #9271, 1:200), and anti-Phospho-Met (Tyr1234/1235) (Cell Signaling Technology, #3077, 1:200).

Protein lysates were made using lysis buffer (20 mM Tris pH 8.0, 300 mM NaCl, 2% NP40, 20% glycerol, 10 mM EDTA) complemented with protease inhibitors (Roche) and quantified using the BCA Protein Assay Kit (Pierce). Protein lysates were loaded onto a 3–8% Tris-Acetate gradient Gel (Invitrogen) and transferred overnight onto PVDF membrane (Millipore, methanol pre-wetted) in transfer buffer (38 mM glycine, 5 mM TRIS and 0.01% SDS in PBS-T (0.5% Tween-20). Membranes were blocked in 5% ELK in TBS-T after which they were stained for four hours at room temperature using the primary antibody anti-Met (1:1000, Cell Signaling 4560S), anti-phosphoMet (1:1000, Cell Signaling 3077S), anti-Myc (1:1000, Abcam ab32072), or anti-Mcl1 (1:1000, Cell Signaling 94296S). Membranes were washed three times with 1% milk in PBS-T and incubated for 1 h with an HRP-conjugated secondary antibody (1:2000, DAKO). Stained membranes were washed three times in 1% milk in PBS-T and developed using SuperSignal ECL (Pierce).