Table 1.
Primary Antibodies used for Western Blot (WB) and Immunohistochemistry (IHC).
Table 2.
Primers used for quantitative real-time RT PCR.
Figure 1.
Protein content and expression of AQP-5 in P2rx7−/− mice lungs.
Immunohistochemical staining of AQP-5, Aqp5 mRNA expression and analysis of AQP-5 protein content in lungs from wild type and P2rx7−/− mice aged (A) 2–3 months and (B) 11–12 months. Scale bar correlates to 50 µm in immunoperoxidase demonstrations. Quantitative real-time RT PCR was done by using Rpl32 and Polr2a as housekeeping genes. Results are shown median with whiskers extending to the minimum and maximum (n(2–3 months) = 18, n(11–12 months) = 9). Equal protein amounts were used in SDS-PAGE and analyzed by western blot with the antibody against AQP-5. Relative protein levels AQP-5/γ-Tub are shown median with whiskers extending to the minimum and maximum (n(2–3 months) = 15, n(11–12 months) = 15) and one representative blot is pictured (* p<0.05).
Figure 2.
Sirius Red staining and immunohistochemical staining of CD44v10 and CC3 in P2rx7−/− mice lungs.
Immunoperoxidase demonstration of Sirius Red (A), CD44v10 (B) and CC3 (C) in lungs from two cohorts of wild type and P2rx7−/− mice (younger age bracket 2–3 months; older age bracket 11–12 months). Scale bar 50 µm.
Figure 3.
Protein content and expression of Cav-1, T1α and Cx43 in P2rx7−/− mice lungs.
mRNA expression, analysis of corresponding protein content and immunoperoxidase demonstration of Cav-1 (A), T1α (B) and Cx43 (C) in lungs from wild type and P2rx7−/− mice aged 2–3 months compared to the particular protein content in wild type and P2rx7−/− mice aged 11–12 months. Quantitative real-time RT PCR was done by using Rpl32 and Polr2a as housekeeping genes. Results are shown median with whiskers extending to the minimum and maximum (n = 18). Equal protein amounts were used in SDS-PAGE and analyzed by western blot with antibodies against the particular protein. Protein levels are normalized to γ-Tub and are shown median with whiskers extending to the minimum and maximum (n(2–3 months) = 15, n(11–12 months) = 10) and one representative blot is pictured. Scale bar correlates to 50 µm in immunoperoxidase demonstrations.
Figure 4.
Collagen and AQP-5 protein content in wild type mice compared to P2rx7−/− knockout mice after BLM treatment.
Immunoperoxidase demonstration of AQP-5 (A) and Sirius Red staining (B) of wild type and P2rx7−/− mice lungs after BLM treatment with 200 mU/ml for 72 h in PCLS. Untreated lung slices after 72 h were used as controls. Scale bar 50 µm. For analysis of AQP-5 protein content, equal protein amounts were used in SDS-PAGE and analyzed by western blot with the antibody against the AQP-5 (C). BLM treated and untreated PCLS from wild type and P2rx7−/− mice lungs were compared statistically. Relative protein levels AQP-5/γ-Tub are shown mean ± SEM (n = 4) and one representative blot is pictured (* p<0.05).
Figure 5.
Dependency of AQP-5 on P2X7R expression.
Analysis of protein content in MLE-12 cells (A) and E10 cells (B) after BLM treatment: Cells were treated with 100 mU/ml BLM, 100 µM oxATP with BLM (100 mU/ml) and 100 µM oxATP for 24 h. oxATP was added 2 h prior to BLM treatment. Untreated cells were used as control. For SDS-Page equal protein amounts of cell lysates were used and analyzed by western blot with antibodies against P2X7R and AQP-5. Protein levels are normalized to γ-Tub and are shown mean ± SEM (n = 6) in relation to control. One representative blot is pictured (* p<0.05).
Figure 6.
AQP-5 and P2X7R expression over the course of time.
Analysis of protein content in MLE-12 cells (A) and E10 cells (B) after BLM treatment: Cells were treated with 100 mU/ml BLM for 24 h, 48 h and 72 h. Untreated cells after 24 h, 48 h and 72 h were used as control. For SDS-Page equal protein amounts of cell lysates were used and analyzed by western blot with antibodies against P2X7R and AQP-5. Protein levels are normalized to γ-Tub and are shown mean ± SEM (n = 3) in relation to control. One representative blot is pictured (* p<0.05).