presence of HS motifs with high affinity, while FGF19 and FGF21 cannot bind directly to the HS-FGFR but in the presence of the transmembrane KLB. It has been shown that the N- and C- terminuses of FGF19 and FGF21 are required for the interaction with FGFR and KLB, respectively; however, it is obvious that the N-terminus alone is not sufficient for direct interaction with FGFR without the presence of KLB [53,54,57]. These imply that the high affinity binding of FGF19 and FGF21 to KLB, which has been shown in a binary complex with FGFR that is ready to be activated by the binding event [4], is likely the first and key promoting step for the subsequent productive complex formation through more interactions with FGFR that produce further higher affinity and more stability. The integration of KLB into the HS- FGFR for transducing the FGF19 and FGF21 stimulation across the plasma membrane is therefore a hallmark event for their metabolic effects. This integration was visualized by the presence of several possible cross-linking bands. It would be interesting to know how the divergence and specificity in the intracellular phosphorylation and selection of signaling relay adaptors are resulted from the extracellular integration. Materials and Methods Ethics Statement All mice were housed in the Program of Animal Resources in the Institute of Biosciences and Technology, and were handled in accordance with the principles and procedure of the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Institute of Biosciences and Technology Institutional Animal Care and Use Committee (IBT IACUC) with a protocol #10022 entitled ‘‘BetaKlotho-FGFR in the liver’’. Expression plasmid constructs Human full-length FGFR1bIIIC (NM_023105) and FGFR4 (AAM13666) in the Tet-on pcDNA4/TO mammalian inducible expression vector, and murine KLB (NM_031180) in pEF1a (a gift from Dr. Kuro-O M) [9] were described previously [4]. Human FGF19 and murine FGF21 in pET28 with 66His tag at the N- terminus were expressed in BL21 DE3 E. coli, and refolded from inclusion body or directly purified from soluble fraction on Ni- chelating Sepharose chromatography using AKTApurifier (GE HealthCare, MA). Recombinant human FGF21 with a N-terminal 66His tag with an activity equal to our preparation of murine FGF21 as determined in our ERK activation assay (Figure 6) was from Dr. Xiaokun Li (Wenzhou Medical College, China). Cell lines The establishment, maintaining and protein expression induc- tion of the tetracycline (Tet)-inducible T-Rex-293 cell lines (Invitrogen, CA) expressing the iFGFR1-cKLB, iFGFR4-cKLB, iFGFR1, iFGFR4 or cKLB were described previously [4]. The generation and maintaining of the HR4 hepatoma cell line was also described in our previous study with FGFR42/2 and DEN hepatoma model [4,70]. Mouse tissues The KLB2/2 mouse line with an inactivating insertion in the first intron was obtained by gene-trap technology (TIGM, Texas). The homozygous C57/BL6 mice exhibited an increase in hepatic bile acids and about 35% less survival rate than expected, similar to that previously reported [60]. Mice deficient in FGFR1 in specifically the adipose tissue (AdiFGFR1) were generated by using FGFR1f/f (Flox) and aP2-Cre mice [71,72] (manuscript in preparation). The aP2 promoter is relatively adipocyte-specific. FGF21 Activates Preferentially Adipose FGFR1-KLB AdiFGFR1 conditional (CN) mice were obtained from FGFR1f/f mice and aP2-Cre+/w mice, and then breeding with C57/BL6 for more than five generations. qPCR results indicate that FGFR1 was reduced to nearly an undetectable level in adipocytes in WAT. The male C57/BL6 mice at 5 or 6 weeks old were provided standard feeding and water ad libitum according to the protocols approved by the Institutional Animal Care and Use Committee. Before injection mice were fasted overnight by withdrawal of food with water still available. Pure recombinant FGF19 or FGF21 at 0.5 mg/Kg or vehicle (16 PBS) were then injected intraperitone- ally into six mice in each group. After 20 min, mice were sacrificed, and the left lobes of the livers, gonadal adipose tissues, hypothalamus, pancreas, skeletal muscle, etc., were isolate, and frozen at 280uC. Quantitative PCR analyses Total RNA was isolated from cells or tissues using Ultraspec RNA Isolation reagents (Biotecx Laboratories, Houston, TX). Equal amounts of total RNA from five mice were pooled and then 5 ug was subjected to the reverse transcription. Primers 59- ACTCCTTCTCCAGCATGGGCTC and 59-AGTTGAATC- TGTCTCCGCTTGGAG were used to quantify the expression level of c-Fos. Primers pairs 59-CTGAAGGAGGGTCATC- GAAT and 59-GTCCAGGTCTTCCACCAACT, 59-CAC- CACGGACAAAGAGATTG and 59-TGTCAACCATGCAGA- GTGAA, 59-AGATGCTGAAAGATGATGCG and 59-AT- GATGTTCTTGTGCTTGCC, and 59-CAGAGGCCTTTGG- TATGGAT and 59-AGGTCTGCCAAATCCTTGTC were for mouse FGFR1, FGFR2, FGFR3 and FGFR4, respectively. Primers 59-CAGAGAAGGAGGAGGTGAGG and 59-CAG- CACCTGCCTTAAGTTGA were for mouse KLB. These primer pairs were designed by the real-time PCR (TagMan) primer design program from GenScript, all with the Tm of 59uC and the amplicon length of 80–150 bp. Relative gene expression was measured by real-time PCR with 40 cycles using the SYBR Green JumpStart Taq Ready Mix (Sigma) on the Stratagene Mx3000P qPCR system. All measurements were done in triplicate, and relative amounts of mRNA were calculated by the comparative threshold (Ct) cycle method using b-actin as the internal control, and presented as mean6S.E.M using two-tailed unpaired Student’s t-test. For the comparison of expression levels of FGFRs and KLB across all different tissues examined, the threshold cycle times were subtracted with 34, which was determined as the cycle limit for detection. Normalized expression levels were expressed as arbitrary folds of difference by dividing by that of b-Actin detected in each sample, and multiplied by 104 for graphic presentation. Data were presented as the mean of triplicates 6 S.D. Immunoblot analyses Cells after treatment were lysed in 16 SDS sample buffer. Alternatively, cells were lysed in modified cold RIPA buffer of 20 mM Tris-HCl, pH 7.2, 50 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 2 mM sodium orthovanadate (pre-oxidized by H2O2 prior to use) and 2 mM NaF, and before use, 1 pill each of protease inhibitors and phosphatase inhibitors (Roche, IN) per 10 ml were added. Supernatants after centrifugation were used to perform immunoprecipitation with antibodies and protein A/G agarose. Whole cell lysate supernatants or immunoprecipitates were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membrane was probed with the antibodies against FGFR4, KLB, ERK and phospho-ERK1/2 (Santa Cruz Biotechnology, CA), and pTyr (Cell Signaling Technology, Inc). For sequential blotting, the wet membranes PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33870