The separation Akt inhibitor of hepatic parenchymal and nonparenchymal cells was performed essentially as previously described.24 The procedure is described in detail in the supporting information. A rat hepatic SEC line (NP31)25 was cultured on type I collagen–coated dishes (Iwaki, Chiba, Japan) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C with 5% CO2. A retrovirus vector (pMxIG)26 and a retrovirus packaging cell line (Plat-E)27 were used to

generate recombinant retroviruses. Hemagglutinin (HA)-tagged complementary DNAs (cDNAs) of full-length Cas (Cas FL) and a Cas mutant lacking the SH3 domain (Cas ΔSH3)28 were subcloned into pMxIG, and ecotropic retroviruses were produced by the transient transfection of Plat-E cells with viral vectors using FuGENE (Roche, Basel, Switzerland). Infection was performed in the presence of 8 μg/mL Polybrene (Sigma, St Louis, MO). After fixation Selleckchem RO4929097 in 4% paraformaldehyde in phosphate-buffered saline for 10 minutes, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 minutes at room temperature and incubated with Alexa 594–conjugated phalloidin (1:40; Invitrogen, Carlsbad, CA) in 1% bovine serum albumin in phosphate-buffered saline for 30 minutes at 37°C. Cells were mounted

with Vectashield 4-Aminobutyrate aminotransferase and observed on an Axioplan2 microscope with AxioCam MRm controlled by Axiovision software (Carl Zeiss, Germany). NP31 cells, cultured on glass cover

slips, were fixed in 2% glutaraldehyde buffered with a 1 M cacodylate buffer (pH 7.4) for 12 hours at 4°C and then with 1% osmium tetroxide in a cacodylate buffer (pH 7.4) for 1 hour at 4°C. After dehydration in a graded series of ethanol solutions, cells were dried to a critical point and sputter-coated with osmium. Cell surfaces were examined with an S-4300 scanning microscope (Hitachi, Tokyo, Japan) at a 30-kV accelerating voltage. To create a reduction-of-function Cas allele by gene targeting, we deleted exon 2 of the Cas gene, which encodes the entire SH3 and the N-terminal part of the SD domain containing one YLVP motif and four YQxP motifs. To this end, we constructed a targeting vector containing Cas exon 2 flanked by two locus of X-over P1 (loxP) sequences and followed by the Frt-flanked neomycin resistance (Neo) gene (Fig. 1A). When the floxed Cas exon 2 was correctly excised, exon 1 joined in frame to exon 3, and this resulted in a Cas transcript devoid of the exon 2–derived segment. Correctly targeted embryonic stem cells, identified by Southern blotting and genomic polymerase chain reaction (PCR; Fig. 1B, left and middle panels), were selected and used for the generation of heterozygous mice (Cas+/floxNeo).