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Semi-quantitative RT-PCR. Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The isolated RNA was converted into cDNAs using Oligo (dT) primers (Invitrogen) and an Omniscript RT Kit (Qiagen). Semi-quantitative RT-PCR was performed for 38 cycles using a PCR Thermal Cycler (TaKaRa). Primer sequences were as follows: PTPRK full-length fwd: 5′-TATAGGCACTGAGGTGCA-3′ and rev: 5′-CTGCTGGCTCAACAGA-3′. The PTPRK gene BMS 232632 levels were normalized by the corresponding gene expression levels of mouse β-actin.
Western blotting. The cells were harvested and lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 0.15 M NaCl, containing 1% NP-40, protease inhibitor cocktail (Roche, Germany), phosphatase inhibitor cocktail 2 (Sigma, St. Louis, USA)). The cell lysates were resolved on SDS–PAGE and transferred to PVDF membranes (Atto, Japan). The membrane was then immunoblotted with each antibody after blocking. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Millipore, Bedford, MA).