Fig 1: Bombesin receptor–activated protein (BRAP) interacts with ATG5 in cultured human bronchial epithelial cell line 16HBE14o.(A) The interaction between full length ATG5 and the CTD of BRAP analyzed by yeast two-hybrid assay. The diploids containing both full length ATG5 and the CTD of BRAP formed white colonies on agar plates containing SD/–Leu/–Trp media. Those diploids growing on agar plates containing SD/–Ade/–His/–Leu/–Trp media turned blue with the presence of X-α-Gal, indicating the physical interaction exists between the full length ATG5 and BRAP. (B) Co-immunoprecipitation analysis of lysates of cultured 16HBE14o- cells using either anti-ATG5 antibody or anti-BRAP antibody to immunoprecipitate cell lysates. 5% of input of cell lysates, control experiments using an anti-actin antibody or an anti-histone antibody, and the immunoprecipitated samples were loaded in every other well on a 10% polyacrylamide gel, and then Western blotting analysis using either the anti-BRAP antibody (upper panel) or the anti-ATG5 antibody (lower panel) were performed to detect signals. (C) Interaction of recombinant GST-tagged ATG5 and MBP-tagged CTD of BRAP by in vitro pull down assay. Glutathione agarose resin was incubated with Escherichia coli cell lysates containing the recombinant GST-tagged ATG5 and then washed thoroughly. The beads was then incubated with E. coli cell lysates containing the MBP-tagged CTD of BRAP and washed to pull down CTD of BRAP. The samples were loaded in every other well on 10% polyacrylamide gel. 10 μl of cell lysates containing recombinant MBP-tagged CTD of BRAP was used as the input sample, which is shown in lane 1. The pull down sample is shown in lane 2. And the control experiment using glutathione agarose resin bound with the GST-tagged ATG5 to incubate with equal volume of PBS instead of cell lysates containing MBP-tagged CTD of BRAP is shown in lane 3. The immunoblot was probed with antibody against BRAP. The molecular weight of recombinant MBP-tagged CTD of BRAP is about 74 kD (the CTD of BRAP is around 32 kD). (D) Representative images of Western blotting analysis of lung fibroblasts isolated from mice. After starvation for 24 h, cells from individual mouse were lysed and then loaded in the wells next to the ones without starvation on 10% polyacrylamide gel (left panel). The right panel shows the quantification of ATG5 protein bands from six independent experiments before and after starvation. n = 6 in each group, *P < 0.05. (E) qRT-PCR analysis of mRNA expression of ATG5 in lung fibroblasts from mice. n = 3.Source data are available for this figure.
Fig 2: Bombesin receptor–activated protein (BRAP) homologous protein deficiency attenuated bleomycin-induced pulmonary inflammation in mice.(A) Representative images of BRAP immunostaining in human tissue sections. Interstitial cells were indicated by red circles (original magnification ×400). Scale bar = 50 µm. (B) Representative images of immunofluorescence staining in cultured human lung fibroblast (HLF) cells. Immunostaining with anti-BRAP antibody (green color) was shown in the upper panel. Nuclei of the cells were localized by DAPI (blue). Double immunofluorescence staining in HLF cells was shown in the lower panel. Immunostaining using BRAP antibody was shown as green fluorescence and immunostaining using S100A4 antibody was shown as red fluorescence. The orange signals in the merged picture indicate the co-distribution of BRAP and S100A4 in the cytoplasm of fibroblasts (original magnification ×400). Bar = 50 µm. (C) BRAP homologous protein was present in lung tissues as revealed by Western blotting in wild-type control mice BC004004+/+ with or without bleomycin treatment. The sample from an individual animal was loaded in one well. There was no BRAP homologous protein detected in lung tissues from BC004004-/- mice. The expression of ß-actin was detected as a loading control. BLM, bleomycin. (D) The body weight changes were shown as percentages of weight gain on Day 21 after bleomycin instillation compared with the body weights on Day 0. Data are presented as mean ± SD; n = 7. *P < 0.05, **P < 0.01. (E) Representative histological changes of lung tissues after bleomycin treatment in both BC004004-/- mice and their wild-type control mice (original magnification ×200). Scale bar = 100 µm. (F) Ashcroft scores were determined for each tissue section (one tissue section from one mouse). Data are presented as mean ± SD; n = 31 for each group. ****P < 0.0001. (G) The total cell numbers in bronchoalveolar lavage fluids (BALF) from mice on Day 21 after bleomycin instillation. There are more cells in BALF from BC004004+/+ mice compared with BC004004-/- mice after bleomycin treatment (*P = 0.0451, n = 4). Data are presented as mean ± SD. n = 4. *P < 0.05, **P < 0.01.Source data are available for this figure.
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