In the present study, we demonstrated that HAb18G selleckchem Sorafenib CD147 interacts with integrin 6 1, activates the PI3K signal pathway through phosphorylation, and thereby enhances the invasion potential of hepatoma cells. Methods Cell culture Human SMMC 7721 and FHCC98 cells were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicil linstreptomycin, and 2% L glutamine at 37 C in a humidified atmosphere of 5% CO2. Gene silencing of CD147 by RNA interference, RT PCR and Western blot The sense sequence for Inhibitors,Modulators,Libraries the HAb18GCD147 small inter fering RNA cells were transfected with siRNA using Lipofectamine 2000 reagent according to the manufacturers instructions. Silencer negative control siRNA was used as a negative control under similar condi tions.

Silencing effects of HAb18GCD147 were examined by RT PCR and Western blot. Forty eight hours after siRNA transfection, total RNA was isolated using Trizol according Inhibitors,Modulators,Libraries to the manufacturers instructions for use in the analysis Inhibitors,Modulators,Libraries of CD147 Inhibitors,Modulators,Libraries mRNA levels. The isolation was fol lowed by first strand cDNA synthesis using True Script reverse transcriptase. The cDNA was amplified by PCR using a specific primer set for CD147 or glyceraldehyde phosphate dehydrogenase as an internal control. The sequences Inhibitors,Modulators,Libraries of the upstream and downstream primers were, respectively, as follows Sense 5 for GAPDH. PCR analysis was performed under the follow ing conditions an initial denaturation at 94 C for 2 min followed by 25 30 cycles of a 30 sec denaturation step at 94 C, renaturation for 60 sec at 57 C, and extension for 90 sec at 72 C.

The amplified products were analyzed by 1% agarose gel electrophoresis followed by GoldView staining. Forty eight hours after siRNA transfection, FHCC98 and 7721 cells were harvested in a lysis buffer and equal amounts of cellular proteins were subjected sellckchem to SDS PAGE. Proteins were transferred to polyvinyli dene difluoride membranes and blots were probed with HAb18 mAb. GAPDH was chosen as an internal control and the blots were probed with mouse anti GAPDH mAb. Co immunoprecipitation and Western blot analyses The interaction of HAb18GCD147 with integrin 6 1 in native cells was detected using the ProFound Mamma lian Co Immunoprecipitation Kit according to the manufacturers instructions. Briefly, FHCC98 cells or 7721 cells were lysed with M per reagent. The lysate was collected onto a coupling gel by four washes with the co immunoprecipitation buffer and which were pre bound with 200g mouse anti human HAb18G CD147 mAbHAb18 or 200g mouse anti human 6 mAb and 200g mouse anti human 1 mAb.