Western blots and Zymography were performed as described.4, 11 Proteins (40 μg/sample) in sodium dodecyl sulfate (SDS)-loading buffer were electrophoresed through 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Membranes were incubated with specific antibodies against cleaved caspase-3 CH5424802 (ASP175), phospho-AKT (D9E; C31E5E), AKT (C67E7), phospho-c-Met (D26 and 130H2), c-Met (25H2) (Cell Signaling),
Bcl-2 (Abcam), and cyclin D1 (BD Biosciences). After development, membranes were stripped and reblotted with antiactin antibody (Santa Cruz). Gelatinolytic activity was detected in liver extracts (100 μg) by 10% SDS-PAGE contained 1 mg/mL of gelatin (Invitrogen) under nonreducing conditions. After incubation PLX3397 molecular weight in development buffer (50 mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02% NaN3, pH 7.5), gels were stained with Coomassie brilliant blue R-250 (Bio-Rad) and destained with methanol/acetic acid/water (20:10:70). Prestained molecular weight markers (Bio-Rad) and MMP-9 (BIOMOL International) served as standards. Relative quantities of protein were determined using a densitometer (NIH Image J software). RNA was extracted from livers with Trizol (Life Technologies) as described.4 Reverse transcription was performed using 5 μg of total RNA in a first-strand
complementary DNA (cDNA) synthesis reaction with SuperScript III RNaseH Reverse Transcriptase (Life Technologies) as recommended by the manufacturer. The cDNA product was amplified find more by PCR using primers specific for each target cDNA.
Data in the text and figures are expressed as mean ± standard deviation. Statistical comparisons between groups of normally distributed data were performed with Student’s t test using statistical package SPSS (Chicago, IL). Kaplan-Meier analysis was used to determine statistical significance of the differences in mouse survival. P < 0.05 was considered statistically significant. TIMP-1 messenger RNA (mRNA) was almost undetectable in naive livers and it was significantly up-regulated in TIMP-1+/+ livers from 3 hours to 7 days postreperfusion (Fig. 1A). TIMP-1 protein expression was mildly detected in TIMP-1+/+ naive livers and it was markedly increased in livers after 6 hours of reperfusion, particularly at 24 hours and 48 hours post-IRI (Fig. 1B). Immunofluorescence analysis showed TIMP-1 staining in the surviving parenchyma predominantly around the portal triads of wildtype livers (Fig. 1C); TIMP-1+ staining was mostly detected in cells along hepatic sinusoids, likely hepatic stellate cells (HSCs), and in scattered hepatocytes. In vitro studies have linked TIMP-1 production to HSC and to hepatocytes.13 Conversely, TIMP-1 staining was absent in TIMP-1−/− livers after IRI (Fig. 1C). To test the significance of TIMP-1 expression in liver IRI, our experiments included TIMP-1-deficient and respective wildtype (TIMP-1+/+) control mice.