The abundance of phosphorylated form was always related to the amount of non-phosphorylated protein assessed on the same membrane after incubation with stripping buffer (Pierce). The activation of the particular transduction pathway was presented as ratio between phosphorylated form and total abundance of the protein, and this ratio in average WT placebo treated animal was arbitrarily set as 1. The experimental procedures were performed on the extensively studied and well characterized C57BL/6JIL-6-/-TMKopf Y27632
strain [20, 21, 22?and?23], and results were compared to the respective wild type C57BL/6J animals. The genotypes were assessed as previously described using standard PCR of DNA isolated from mouse tails . Results were presented as the mean �� standard deviation (SD). The student t-test was used for statistical analysis. A p value less than 0.05 was considered statistically Fleroxacin
significant. After placebo injection STAT3 phosphorylation is significantly more pronounced in WT animals as compared to IL-6 KO mice (p < 0.05, t-test) (Figure 1A?and?Figure 1B). Single intraperitoneal injection of isoproterenol results in rapid and potent (more than 3-fold) enhancement of STAT3 phosphorylation in the myocardium of both WT and IL-6 KO animals (p < 0.01, t-test) (Figure 1A?and?Figure CHIR99021
1B). After 24 hours the extent of STAT 3 phosphorylation returns to the levels close to the initial state both in WT and IL-6 KO. The assessed knockout did not influence the activation of MEK/ERK pathway (the extent of ERK 1 and ERK2 phosphorylation in relation to the total ERK 1 and ERK2 protein) after placebo injection. Isoproterenol delivery caused rapid and potent (approximately 10 fold) (p < 0.005, t-test in comparison to the placebo) enhancement of ERK1 and ERK2 phosphorylation in both genotypes (Figure 2A?and?Figure 2B.). The ERK phosphorylation returned to the placebo injection level by the 24 hours mark (Figure 2A?and?Figure 2B.). Three-day treatment with isoproterenol produced consistent hypertrophy of both right and left ventricles (Fig. 3.) (p < 0.05, t-test isoproterenol treated vs placebo treated). A similar increase of ratios between the weights of the ventricles and the length of tibia was observed in both WT and IL-6 KO animals (Fig. 3.). This finding was later confirmed by the analysis of the cardiomyocyte crossectional area (CSA). There were no significant differences in CSA between genotypes among animals that received isoproterenol (Fig. 4.). This paper confirms previous reports that single intraperitoneal injection of isoproterenol enhances STAT3 phosphorylation in myocardium .