RNA isolation and real-time PCR Total RNA was extracted from cells and tissues using RNeasy kits accord- ing to the manufacturer’s instructions (QIAGEN). First-stand cDNA was synthesized using 500 ng of total RNA with 200 U of Superscript II RNase H-RT (Invitrogen) in a ﬁnal volume of 20 μl. The resulting products were then treated with RNase A for 30 min at 37°C and puriﬁed thereafter with Qiaquick PCR puriﬁcation kit (QIAGEN). Real-time PCR was performed using the 7500 Real-time PCR system (Applied Biosystems). Lung histology Mouse tissues were ﬁxed overnight in 4% paraformaldehyde/PBS and pro- cessed for parafﬁn embedding. Deparafﬁnized sections (5 μm) were treated with Antigen unmasking reagent (Vector Laboratories) for 15 min for antigen retrieval. Blocking was achieved with 2.5% horse serum in PBS for 10 min. Primary antibody (anti-GFP and anti–prosurfactant C) incuba- tions were performed for 30 min at room temperature. Biotinylated second- ary antibodies (Vector Laboratories) were added to sections for 10 min, followed by signal detection using NovaRED reagent (Vector Laboratories). Anti–prosurfactant C antibody was a gift from J. Whitsett (Cincinnati Chil- dren’s Hospital Medical Center, Cincinnati, OH). TUNEL assay TUNEL assay was performed according to the manufacturer’s instructions (Promega). In brief, paraformaldehyde-ﬁxed and parafﬁn-embedding lung tissue sections were stained with ﬂuorescein-12-dUTP (Promega). Images were acquired using a confocal microscope (Carl Zeiss, Inc.). Target deletion of mouse BMPER gene Mice deﬁcient for Bmper were generated with standard gene targeting methods (Dai et al., 2003). The targeting construct was generated using pOSfrt (Caron and Smithies, 2001) as the backbone, which contains (a) a 4.4-kb PCR-generated fragment from genomic DNA that includes the BMPER promoter; (b) a 700-bp cDNA encoding EGFP (Clontech Laboratory Inc.); (c) a 300-bp bovine growth hormone poly(A) addition region; and (d) a 2.0-kb PCR-generated genomic fragment from second intron of the BMPER gene. Embryonic stem (ES) cells (129/ola) were electroporated with the tar- get construct. ES colonies that were G418/ganciclovir resistant were identi- ﬁed by the PCR-based assay. Homologous recombination was conﬁrmed by Southern analysis using both 5 and 3 diagnostic probes. The targeted ES cells were injected into C57BL/6 blastocysts (Dai et al., 2003). Male chime- ras were mated to wild-type C57BL/6 females to establish an isogenic line, and all experiments were conducted on the resulting hybrid background. Genotyping of Bmper mutant mice Genomic DNA of Bmper-deﬁcient mice was isolated and digested with HpaI (5 probe) and NheI (3 probe) to identify wild-type and targeted alleles. Bmper homologous mutants were generated by timed hetero- zygous matings. A PCR analysis has been developed to genotype the embryos and pups. PCR samples were denatured in 95°C for 60 s, and then subjected to 35 cycles of three-step ampliﬁcation, a 30-s 94°C denaturation, 30-s 68°C annealing, and 45-s 72°C extension step. A 608-bp product (primers berwtf and bergtr) represents the wild-type allele and a 455-bp product (primers berkof and bergtr) indicates the target allele. PCR primers: berwtf, 5 -CTGCATCCACCC- CTGTAAGTTTCTAG-3 ; berkof, 5 -gtcctctgatggtcaaagtcctg-3 ; bergtr, 5 -CCAAGCCCAACGCTCCCTGCTGAAATCC-3 . Southern blotting analysis Southern blotting analysis was performed as previously described (Matzuk et al., 1992). Genomic DNA was digested with either HpaI or NheI and electrophoresed in a 1% agarose gel. Both 5 and 3 probe were labeled with -[32P]deoxycytidine triphosphate by random oligopriming. Mem- branes containing DNA have hybridized with labeled probes in 65°C for 1 h. The locations of radioactive probe hybridization on membrane were detected by autoradiography. Statistical methods Means ± SEM and a one-way analysis of variance were used for all ﬁgures that required statistical measurements. A multiple comparison procedure was used, using the Tukey method to determine statistical signiﬁcance be- tween groups. Online supplemental material Fig. S1 support endocytic studies in Fig. 3 through acid wash and Bmp4 neutralization experiments. Fig. S2 shows the strategy for deleting 608 JCB • VOLUME 184 • NUMBER 4 • 2009 Bmper in ES cells and in mice. Figure S3 shows the immunohistochem- istry for GFP to support the Bmper phenotype in mice lung from Fig. 7. Online supplemental material is available at http://www.jcb .org/cgi/content/full/jcb.200808064/DC1. 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