Results: 5

1.
Fig. 3.

Fig. 3. From: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Effects of Timp-1, Timp-2, and Timp-3 on KL shedding. (A) COS-7 cells were transfected with KL alone (KL control) or cotransfected with KL and Timp1, Timp-2, or Timp-3 as indicated. Forty-eight hours after transfection, the cells were washed and incubated in serum-free medium as described before. The protein samples in the cell lysates and medium were collected and analyzed by Western blotting. BSA and tubulin were used as loading controls. The expression of Timp-1 was analyzed by Western blotting with anti-Timp-1 antibody, and the expressions of Timp-2 and Timp-3 were analyzed with an anti-V5 antibody. The apparent molecular masses of the bands are indicated. Statistical analysis of the densitometric results is shown in SI Text. (B) Forty-eight hours after transfection, KL-transfected COS-7 cells were incubated in serum-free DMEM without (KL control) or with various amounts of the Timp-3 peptide, as indicated. The proteins in the cell lysates and medium were analyzed as described in Fig. 1.

Ci-Di Chen, et al. Proc Natl Acad Sci U S A. 2007 December 11;104(50):19796-19801.
2.
Fig. 5.

Fig. 5. From: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Effect of Insulin on ADAM10 and ADAM17 activities, mRNA level, and KL shedding in COS cells. (A) KL-transfected COS-7 cells were treated without (Control) or with (+Insulin) 1 μM insulin for 2 h by using the same sample preparation and analysis procedures as described in Fig. 1. The blots were analyzed with the indicated antibodies. BSA and tubulin were used as loading controls. The ADAM10 and ADAM17 proteins (arrowheads 1 and 2, respectively), and a secreted derivative of APP (arrowhead sAPPα) are indicated. (B) RT-PCR of specific genes. Total RNA was purified from transfected cells with no insulin treatment (−) or with insulin treatment (+) as described in A for the genes indicated. Statistical analysis of the results are shown in SI Text.

Ci-Di Chen, et al. Proc Natl Acad Sci U S A. 2007 December 11;104(50):19796-19801.
3.
Fig. 2.

Fig. 2. From: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Regulation of KL shedding by insulin signaling pathway. (A) KL-transfected COS-7 cells were treated without (Control, lanes 1–3) or with (lanes 4–7) 1 μM insulin in serum-free DMEM for the times indicated. In lane 7, the cells were treated with 100 nM wortmannin, the insulin pathway inhibitor, for 15 min before insulin treatment for 120 min (Wort + Insulin). The proteins in the medium and cells were analyzed as described in Fig. 1. BSA and tubulin were used as loading controls. (B) Densitometric analysis of the intensity of the 130- and 68-kDa bands obtained from Western blotting from control (Con), insulin-treated (Ins), and wortmaninn-plus-insulin-treated (Wort) for 120 min from three independent transfections (for typical results, see lanes 3, 6, and 7 in A). The intensity of each band was normalized to the intensities of BSA or tubulin from each sample. The relative percentage using the average intensity of the 130-kDa band in control as 100% was analyzed and plotted. Error bars represent SD of the relative percentage (n = 3). *, P < 0.05; **, P < 0.005.

Ci-Di Chen, et al. Proc Natl Acad Sci U S A. 2007 December 11;104(50):19796-19801.
4.
Fig. 4.

Fig. 4. From: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

ADAM10 and ADAM17 are responsible for KL shedding in COS-7 cells. (A) COS-7 cells were cotransfected with KL, with empty vector, or with either ADAM10 or ADAM17 as indicated. Forty-eight hours after transfection, the cells were washed and incubated in serum-free medium as described before. The protein samples in the cell lysates and medium were collected and analyzed by Western blotting. BSA and tubulin were used as loading controls. The expression of ADAM17 was analyzed by Western blotting with anti-V5 antibody, and the expression of ADAM10 was analyzed with an anti-HA antibody. Note that the cotransfection of KL with ADAM10 and ADAM17 resulted in increased KL fragments in cells and medium (lanes 3, 5, and 6; asterisks). This particular exposure was chosen to emphasize the difference in KL fragments intensities with cotransfection of KL with either ADAM10 or ADAM17. (B) Comparison of the processing pattern of KL and KL GFP construct cotransfected with ADAM10 or ADAM17. The blots were analyzed with anti-GFP antibody as indicated. The estimated molecular masses of the KL fragments are shown. (C) Schematic diagram of the KL GFP construct. The anti-GFP antibody recognition site is indicated. The α- and β-cleavage sites and the estimated molecular masses of the KL GFP fragments are illustrated. (D) Western blots of samples from COS-7 cells transfected with KL with control siRNA (Control) or with KL and siRNA specific to either ADAM10 or ADAM17 were analyzed as described before using antibodies to KL, ADAM10, and ADAM17. Tubulin and BSA were used as loading controls. ADAM10 (arrowhead 1) and ADAM17 (arrowhead 2) protein are indicated. Statistical analysis of the results from D is shown in SI Text.

Ci-Di Chen, et al. Proc Natl Acad Sci U S A. 2007 December 11;104(50):19796-19801.
5.
Fig. 1.

Fig. 1. From: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Searching for the regulators affecting KL shedding in COS-7 cells and ex vivo. (A) Western blots from COS-7 cells transiently transfected with no DNA control (Mock, lane 1) or with the KL plasmid (lanes 2–13). After exposure to various inhibitors in serum-free DMEM (shown above blot), protein samples were collected from either the cell lysate or medium. BSA is shown as the TCA precipitation loading control. COS-7 cells do not express detectable levels of endogenous KL (lane 1). Exogenous KL expressed as a doublet of 135- and 120-kDa bands (lanes 2–7, asterisks). The blot was slightly overexposed to detect all of the KL processing fragments in the cells. At lower exposure of the blot, we could clearly see the doublet, similarly to that seen in G. The estimated molecular masses of the KL fragments in the medium are indicated (130 and 68 kDa, arrowheads). (B) After KL transfection, COS-7 cells were treated without (Control) or with various concentrations of EGTA and EDTA, as indicated, in serum-free DMEM. BSA and tubulin were used as loading controls for protein precipitation from the medium and total protein concentration in the cell lysates. (C) Schematic diagram of the KL protein structure and its processing in COS-7 cells. KL protein contains a signal sequence (SS), two homologous domains (KL1 and KL2), a transmembrane domain (TM), and a short cytoplasmic domain (CD). The anti-KL antibody recognition region is indicated. The α- and β-cleavage sites and the estimated molecular mass of the KL fragments are illustrated. KL980 indicates the truncated form of KL with amino acids 1–980, in which the TM and CDs of KL have been removed. (D) Comparison of the processing pattern of KL and KL980 lacking the TM region. The arrow indicates that the 68-kDa fragment is not detectable in the cell lysate (lane 2) and barely detectable in the medium (lane 4). (E) Expression pattern of KL in mouse brain and kidney tissues. Both full-length KL (arrowhead) and KL fragments (brackets) similar to the processing pattern in COS-7 cells were found in mouse kidney tissue. The ≈30-kDa band is a breakdown product of KL, and we also see it reproducibly in human CSF (data not shown). (F) Rat kidney slices were treated without (Control) or with 5 μM insulin or 100 μM TAPI-1 in serum-free DMEM for 4 h. The KL in the medium and the tissue was analyzed by Western blotting. Note that full-length KL appears as a doublet in the tissue (135 and 120 kDa), and a single band of a 130-kDa KL fragment was detected in the medium. The 130- and 68-kDa KL fragments are indicated (arrows). Tubulin and a protein in the medium were used as loading controls. (G) Statistical analysis of the results from F. The intensities of the 130- and 68-kDa bands were analyzed and normalized to the KL bands from the tissue lysate by using the average intensity of the controls as 100% from three to four independent experiments. The SD is calculated by using the nonbiased or n − 1 method. Significance of results was found by using Student's t test. *, P < 0.05; **, P < 0.005.

Ci-Di Chen, et al. Proc Natl Acad Sci U S A. 2007 December 11;104(50):19796-19801.

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