Figure 3. In vivo inducible expression of the Sftpc^C121G mutation in adult mice causes an ER-retained SP-C pro-protein.

(A) Strategy for generation of tamoxifen-inducible mice in which tamoxifen treatment of the Sftpc^C121G/C121G R26^Cre line results in removal of an inhibitory intronic PGK-neo cassette. (B) qRT-PCR analysis for Sftpc expression in purified AT2 cells from homozygous Sftpc^{C121Gneo/C121Gneo} and Sftpc^C121G/C121G R26^Cre mice at 7 days after treatment with tamoxifen. Data normalized to 18S RNA are expressed as Sftpc mRNA as a fraction of Sftpc^WT R26^Cre mice. (C) Western blotting of BALF large aggregate fraction from Sftpc^C121G/C121G R26^Cre and Sftpc^WT R26^Cre mice on day 7 after tamoxifen showing the absence of mature SP-C (mSP-C) in the Sftpc^C121G/C121G R26^Cre mice. (D) Western blotting of AT2 cell lysates from Sftpc^WT R26^Cre or Sftpc^C121G/C121G R26^Cre mice 7 days after tamoxifen shows Sftpc^C121G/C121G R26^Cre AT2 cells with an ER retained pro-protein (arrowhead) without posttranslational palmitoylation (arrow) or processing intermediates (brackets) observed in Sftpc^WT R26^Cre AT2 cells. (E) Double-label immunofluorescence staining of whole lung sections for proSP-C (red) and the ER marker KDEL demonstrates reticular proSP-C staining with significant colocalization with KDEL in Sftpc^C121G/C121G R26^Cre mice, compared with the punctate pattern of proSP-C staining distinct from KDEL observed in the Sftpc^WT R26^Cre mice (original magnification, ×60).