Methods Plasmid vectors Eukaryotic expression vectors containing

Methods Plasmid vectors Eukaryotic expression vectors containing the full human SFTPC gene fused to either EGFP tag or hemagglutinin tag were obtained as previously described. I73T point mutation was introduced into the wild type SFTPC gene in these vectors using the QuikChange site directed mutagenesis kit following either the recommended protocol. The successful mutagenesis was confirmed by DNA sequencing. MLE 12 cell lines and transfection The mouse MLE 12 lung epithelial cell line was obtained from the American Type Culture Col lection and maintained in RPMI medium sup plemented with 10% FBS. Cells were transfected using FuGene 6 according to the manufacturers protocol. Stable transfection of MLE 12 cells with pcDNA3 HA hSP C1 197 and pcDNA3 HA hSP CI73T vectors was obtained by selecting transfected cells in the presence of 600 ug ml G418 in RPMI med ium for four weeks.

For drug exposure experiments stable cells were grown 24 hours in the presence of 10 uM of cyclophosphamide, azathioprine, methylpredniso lone or hydroxychloroquine. Immunoblotting Total cell proteins were obtained by lysing the cells in lysis buffer, protease inhibitor. For immuno blotting 30 ug protein were GSK-3 separated under reducing conditions using 10% NuPage Bis Tris and transferred to a PVDF mem brane. The following primary antibodies were used, monoclonal rat anti HA tag, monoclo nal mouse anti GFP and polyclonal goat anti calnexin, polyclonal goat anti calreticulin, monoclonal mouse anti HSP90a b, polyclonal goat anti HSP70 and monoclonal anti b actin HRP conjugate.

Signal was detected using chemiluminiscent labeling with Amersham ECL Detection Reagents, densitometrically quantified and normalized to the b actin signal. Immunofluorescence 24 hours after transfection cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilised with 10% Triton X 100, blocked 30 min in PBS with 5% FBS. The following primary antibodies were used and all in 1,200 dilution, polyclonal rabbit anti mouse LAMP3, monoclonal mouse anti human CD63 LAMP3, polyclonal rabbit anti EEA1, mono clonal mouse anti ubiquitin and polyclonal rabbit anti syntaxin 2. Species specific Alexa Fluor 488 or Alexa Fluor 555 secondary antibodies were used at 1,200. Samples were mounted and Alexa Fluor or GFP fluorescence was examined with Axiovert 135 fluorescent microscope and evaluated with AxioVi sion 4.

7. 1 software. For semi quantitative assessment of colocalization, high magnifica tion confocal microscope images were used. On 14 to 27 different coverslips at least 100 vesicles stained for SP C inhibitor Navitoclax and or syntaxin 2 were counted in a blinded fashion and the percentage of vesicles showing staining for both mar kers was calculated. Similarly, the percentage of vesicles stained for SP C and EEA 1 was calculated.

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