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. 2010 Jan 4;285(16):11998–12010. doi: 10.1074/jbc.M109.046243

FIGURE 2.

FIGURE 2.

Western blot analysis of surfactant protein expression in the lung of hTG mice. A, hSP-A expression in the lung of hTG mice. Total protein (100 μg/lane) from lung tissues of hTG mice and SP-A KO mice or BAL protein (15 μg per lane) from hTG mice were subjected to gel electrophoresis (10% SDS-PAGE for lung tissues and 4–20% gradient SDS-PAGE for BAL fluid) under reducing conditions. The protein in the gel was transferred onto a polyvinylidene difluoride membrane, and SP-A was detected using a rabbit antibody (IgG) to human SP-A at a 1:2000 dilution and then a goat anti-rabbit IgG (horseradish peroxidase-conjugated) antibody. The blot was exposed to XAR film following enhanced chemiluminescent detection. Lung tissues were prepared from the following mice: two SP-A KO (lanes 2 and 3), two SP-A2(1A3) hTG mice (lanes 4 and 5), and two SP-A1(6A4) hTG mice (lanes 6 and 7). BAL fluids were isolated from the hTG mice carrying a single human SP-A transgene, i.e. SP-A1(6A4) (lane 8), SP-A2(1A3) (lane 9), or both human SP-A1 and SP-A2 transgenes, SP-A2/SP-A1(1A3/6A4) (lane 10) and from the control KO mouse (lane 11). Two bands with nearly equal intensity were observed at the position of monomeric SP-A in the BAL from the hTG mice carrying human SP-A1 and SP-A2 transgenes (SP-A2/SP-A1(1A3/6A4) (lane 10)). The band with the slightly larger size is SP-A1 (marked by an asterisk), and the other is SP-A2 (marked by a filled circle). All hTG mice were generated in the SP-A KO background. Purified human SP-A protein (lane 1) used as positive control was from the BAL of an alveolar proteinosis patient. Monomer and dimer of SP-A are pointed out by arrows on the right. B, SP-B, SP-C, and SP-D expression in the lung of hTG mice. Total BAL proteins (15 μg/lane) from hTG mice and SP-A KO mice were subjected to gel electrophoresis (4–15% gradient SDS-PAGE) under reducing conditions. The protein in the gel was transferred onto a polyvinylidene difluoride membrane; SP-B, SP-C, and SP-D were detected by SP-B, SP-C, and SP-D antibody, respectively. The blot was exposed to XAR film following enhanced chemiluminescent detection. No significant differences were observed in the levels of each SP-B, SP-C, and SP-D among hTG mice and KO mice.