Total RNA was extracted in accordance with the procedures outlined in the Trizol product manual. The reverse transcription kit was used to perform the reverse transcription reaction. The first strand cDNA was synthesized selleck chem according to the product manual and stored at ?20 C for future use. The HtrA1 and GAPDH primers were synthesized by the Shanghai Invitrogen Biotechnology Company, dis solved in ddH2O and stored at ?20 C for future use. One microliter of cDNA was added to a 25 ul PCR reac tion. The amplification conditions consisted of an initial denaturation for 5 min at 94 C followed by 30 cycles of amplification with denaturation for 30 s at 94 C, anneal ing for 30 s at 56 C, extension for 30 s at 72 C and a final extension for 10 min at 72 C. PCR products were verified by electrophoresis on a 1.

5% agarose gel. The relative HtrA1 mRNA content was determined by the ratio of HtrA1 to GAPDH intensity, which was calcu lated using the QuantityOne software. Western blotting Inhibitors,Modulators,Libraries Fifty milligrams of tissue sample that had been frozen in liquid Inhibitors,Modulators,Libraries nitrogen was ground and homogenized in 1 ml of RIPA lysis buffer. The homogenate was transferred to a 1. 5 ml centrifuge tube and centrifuged at 16,000 g for 30 min. The concentration of the total protein in the supernatant was measured using the BCA Protein Assay Kit. Ten micrograms of total protein from each esophageal Inhibitors,Modulators,Libraries cancer case was mixed together and used as a single combined esophageal cancer tissue protein sample. The same was done to create the adjacent normal esophageal protein sample.

A polyacrylamide gel consisting of a 5% stacking gel and a Inhibitors,Modulators,Libraries 12% separating gel was cast. A Inhibitors,Modulators,Libraries total of 50 ug of protein was loaded per lane, separated by electrophoresis and transferred to a PVDF membrane via the wet trans fer method. After blocking in a solution of 5% non fat milk in TBST at room temperature for 1 h, a rabbit polyclonal anti human HtrA1 antibody or a mouse anti human B actin mono clonal antibody was applied to the blot and incubated at 4 C overnight. The appropriate IRDye 800 labeled secondary antibody was added and incubated at 4 C overnight. After wash ing with TBST, the membrane was scanned with the Odyssey Infrared Imaging System. The relative HtrA1 protein content was determined using the ratio of HtrA1 intensity to B actin intensity, which was analyzed using the QuantityOne software.

Immunohistochemistry The 4% paraformaldehyde fixed tumor tissue was paraf fin embedded, cut into 5 um serial sections and blocked 17-AAG Tanespimycin with normal goat serum at room temperature for 20 min. The tissue section was probed with the HtrA1 antibody at 4 C over night and then washed three times with PBS for 2 min each. The section was subsequently probed with the bio tinylated goat anti rabbit IgG secondary antibody at 4 C overnight and washed three times with PBS for 2 min.