Anti sip1

Protein preparations from the indicated cells were obtained by lysing samples in 50 mmol/L of TRIS (pH 7.5), 100 mmol/L of NaCl, 1% NP40, 0.1% Triton, 2 mmol/L of EDTA, 10 Ag/mL of aprotinin, and 100 Ag/mL of phenylmethylsulfonyl-fluoride. Prestained molecular weight standards were from Bio-Rad (Milan). Proteins separated on the polyacrylamide gels were blotted on a PVDF membrane. The membrane was stained with Ponceau S (Sigma) to enable us to evaluate the success of transfer, and to locate the molecular weight markers. Free protein binding sites on the PVDF membrane were blocked with nonfat dry milk and a Tween 20/TBS solution. The membranes were washed and stained with specific primary antibodies and with secondary antisera, and then conjugated with horseradish peroxidase (Sigma-Aldrich) diluted 1:2,000. The anti-E-cadherin, anti-N-cadherin, anti-CXCR2, anti-phospho-P38, anti-P38, anti-IκB, anti-NF-κB, anti-slug, anti-twist1, anti-ZEB1, anti-Snail and anti-SIP1 antibodies were purchased from Abcam, and the anti-GRO-α antibody was from Santa Cruz. The luminescent signal was visualized with the ECL Western blotting detection reagent kit (Amersham) and quantified by scanning with a Discover Pharmacia scanner equipped with a Sun Spark Classic Workstation.