Figure 5
Protein expression and phosphorylation of Tie2 receptor were not influence by VT treatment 30 h p.i. Mice were intranasally infected with S. pneumoniae (S. pn; 5 × 106 colony-forming units per mouse) or sham-infected as control with phosphate buffered saline ((PBS) + hyaluronidase). Twenty-two h after infection, Vasculotide (VT) (500 ng/100 µl i.v.) or PBS, and ampicillin (0.4 mg/kg bw i.p.) or 0.9% saline were administered and 24 h post infection (p.i.) mice were ventilated for 6 h. A second dose of VT together with 1 mg human serum albumin (HSA) was administered 1.5 h prior to finishing the experiment. (A) To assess Tie2-phosphorylation, proteins were isolated from lung homogenates and Western blot was performed with antibodies for phospho-Tie2 (pTie2), Tie2 (staining after incubation with stripping buffer for detaching the pTie2 antibody from the membranes) and actin as loading control, and developed by infrared imagine. Representative blots for all groups are shown: Membrane 1 (left images) for the non-infected groups, membrane two (right images) for the infected groups. The black line separates the two membranes, dotted lines show the areas where the membranes were cut in two sections before staining. After staining both sections were developed together by infrared imagine. For quantification, the ratio between Tie2 and actin densitometry (B) and pTie2 and actin densitometry (C) was calculated, respectively. The quotient of pTie2 to Tie2 (D) was used to estimate the phosphorylation status of the receptor. Values are listed as median ± IQR with minimum/maximum values, individual values are shown as dots (control n = 4, all other groups n = 8–9, Mann–Whitney-U-Test).
