Figure 1: Expression of MG53 in lung tissue.

(a) Homogenates of lung tissue derived from the wild-type and mg53−/− mice were used for western blotting for the detection of MG53. rhMG53 (0.1 ng) was used as positive control. For comparative purpose, the content of MG53 in skeletal muscle was assayed at different concentrations of muscle tissues. Ponceau S staining reveals differential loading of the skeletal muscle and lung tissues. A nonspecific 40-kDa protein was also recognized by our custom-made anti-MG53 antibody. The uncropped western blotting images are shown in Supplementary Fig 1. (b) Characterization of lung mg53 transcripts by RACE cDNA amplification. The cDNA amplification strategy is illustrated in the upper panel. A mouse lung cDNA preparation was amplified using AP1 and MA1 primers in the 5′-RACE reaction or using MS1 and AP1 primers in the 3′-RACE reaction. Amplified cDNA products in each RACE reaction were analysed in agarose electrophoresis as shown in the lower panel. Putative full-length cDNAs marked with asterisks were extracted from agarose gels and subcloned into a plasmid vector for sequencing. The protein-coding sequence of the amplified lung mg53 cDNAs was identical to that of muscle mg53 cDNAs determined in our previous study. (c) Immunohistochemical (IHC) staining with an anti-MG53 antibody revealed high level of MG53 in wild-type skeletal muscle, which is absent in the mg53−/− muscle. (d–f) IHC staining of lung tissues derived from wild-type (left panels) or mg53−/− mice (right panels). Compared with skeletal muscle, low level of MG53 could be detected in the lung (d). Background staining in the mg53−/− lung probably reflects autofluorescence of capillary cells or nonspecific activity of the anti-MG53 antibody (see the 40-kDa band in a). The MG53 expression pattern in wild-type lung matches to that of AT1α, a type I alveolar cell marker (e). The MG53 and AT1α (type 1a angiotensin II receptor) (anti-AT1α, Novus Biologicals NB600-1015) stainings (d,e) were shown separately to better indicate the localization of MG53 due to its low expression in alveolar cells. The cross-section of bronchioles revealed negative staining for MG53 in both airway smooth muscle layer (arrow) and the neighbouring ciliated endothelial lining of the airway lumen (arrow head) (f). Scale bars, 50 μm. (g) Immunoblotting of MG53 with lysates from mouse lung tissues, and cells derived from A549, human pulmonary artery endothelial cell (PPAEC), human lung microvascular endothelial cell (MVEC-L) and human umbilical vein endothelial cell (HUVEC; 50 μg per lane). rhMG5 (1 ng) served as a positive control for the immunoblotting.