Quantitative real-time PCR For detection of ADAM10 mRNA expression we used SYBR Green I intercalating dye and for ADAM17 and b-actin mRNA expression TaqMan quantitative real-time PCR. 2.5 ml of the respective cDNA were amplified with the AmpliTaq Gold GeneAmp 5000 10 x PCR Buffer II & MgCl2 Solution Kit or the Power SYBR Green Master Mix (Applied Biosystems by Life Technologies, Darmstadt, Germany). The following experimental protocol was applied for both SYBR Green I and TaqMan assays. PCR reaction (40 cycles) was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems): initial denatur- ation for 10 minutes at 95uC, followed by 40 amplification cycles at 95uC (15 seconds), annealing at 60uC (1 min). To confirm product specificity melting curves were performed to verify that only the specific product was synthesized and to exclude the formation of primer dimers. The specificity of the PCR was further verified by subjecting the amplification products to agarose gel electrophoresis. Plasmids containing ADAM10, ADAM17 or b- actin were applied to produce standard curves which were used to calculate the exact copy number of each mRNA. Therefore the plasmid DNA was diluted in 10-fold serial dilutions (107–102 copies) and amplified along with cDNA samples. The copy numbers of the specific target genes were normalized to 106 copies of the housekeeping gene b-actin. Immunodetection After incubation with the respective substances the transfected cells were washed twice with PBS and were lysed in the respective lysis buffer. Equal amounts of protein were subjected to SDS- PAGE under reducing (cleaved caspase-3, cleaved PARP, ADAM10, pSTAT3, PDI, CHOP and BiP) or non reducing (ADAM17) conditions using Tris-Glycine gels (7.5–15%). The proteins were then transferred onto PVDF (Polyvinylidene Fluoride) (ADAM10 and ADAM17) or nitrocellulose membranes (cleaved caspase-3, cleaved PARP, pSTAT3, PDI, CHOP and BiP). After the transfer the membranes were blocked in 5% non-fat dried milk powder in PBS, 0.3% Tween20 and 0.05% Triton X- 100 and further incubated with the respective primary antibody (1:1,000–3,000) in PBS containing 1–5% (w/v) non-fat dried milk powder or 5% (w/v) BSA, 0.3% Tween and 0.05 % Triton X-100 at 4uC overnight. Anti-cleaved caspase-3, anti-cleaved PARP, anti- BiP, anti-PDI (Cell Signaling Technology, New England Biolabs, Frankfurt, Germany), anti-CHOP (Santa Cruz Biotechnology, Heidelberg, Germany), anti-ADAM10 (Sigma Aldrich, Munich, Germany), anti-ADAM17 (R&D Systems, Minneapolis, MN) and anti-GAPDH (Fitzgerald, Concord, MA) were used as primary antibodies. For detection of P-STAT3 and total STAT3 PathScan P-STAT3 (Tyr705), PathScan total STAT3 ELISA (Cell Signaling Technology) and PhosphoPlus STAT3 (Tyr705) Antibody Kit were used according to the manufacturer’s instructions. 125I-leptin binding assay The culture medium was aspirated and the cells were incubated in 1 ml Krebs-Ringer buffer supplemented with 0.5% BSA (w/v) per 6-well for 30 min at 37uC and 5% CO2. Subsequently 0.5 kBq leptin tracer (Mediagnost, Reutlingen, Germany) were added per well and the cells were incubated for 1 h at 37uC and 5% CO2 in the presence or absence of 3 mg/ml unlabeled leptin. Cells were washed twice with PBS/0.5% BSA (w/v) and lysed with 1 ml 0.1 M NaOH solution. Activity in the cell lysates was determined by gamma counter measurements (Berthold LB2111, Berthold Technologies, Bad Wildbad, Germany). Specific binding was determined by subtracting the radioactivity bound in the presence Regulatory Mechanisms for sOb-R Generation of 3 mg/ml unlabeled leptin (nonspecific binding) from the radioactivity bound in the absence of 3 mg/ml unlabeled leptin. Detection of sOb-R sOb-R in the supernatant of transfected cells was detected with an in-house immunofunctional assay as described previously [15]. The in-house immunofunctional assay was also used for determination of Ob-R protein expression in whole cell lysates. The cell lysates were diluted 1:5 in PBS before they were applied in the assay. Statistical analysis Data are presented as means 6 SD. For statistical analysis, we used the STATISTICA 6.0 software program. In the case of non- normally distributed data, non-parametric statistical tests were used for all statistical analyses. Otherwise parametric tests were applied. A P-value ,0.05, was considered statistically significant. Results Constitutive Ob-R shedding and mediating proteases Human membrane-anchored Ob-R isoforms are substrates for constitutive Ob-R cleavage. Although it is known that sOb-R is generated through cleavage of membrane- anchored receptors, is it not yet clear which isoforms serve as substrate and which protease(s) mediate(s) the cleavage process. So we transfected HEK cells with the long (Ob-Rfl) or one of the three short Ob-R isoforms (Ob-R 219.1, Ob-R 219.2 and Ob-R 219.3) to determine if sOb-R is constitutively generated from these four receptor isoforms. After 48 h incubation at 5% CO2 and 37uC the cell supernatants were collected. Fig. 1A shows that all isoforms could serve as substrate for the shedding process. 125I- leptin binding assays and determination of Ob-R protein expression in whole cell lysates were performed as expression and functionality control for the different Ob-R isoforms. Fig. 1 B and C illustrate that short Ob-R isoforms 219.2 and 219.3 showed the highest Ob-R protein-expression in whole cell lysates as well as the highest leptin binding. When the amount of generated sOb-R was normalized to the membrane expression of the respective isoform, determined by 125I-leptin binding, there was a trend for an inverse correlation between the length of the cytoplasmic domain and the amount of shed Ob-R (Fig. 1 D). Similar results were obtained through normalization of sOb-R to Ob-R protein in whole cell lysates of HEK cells transfected with the respective Ob-R isoforms (Supporting information Fig. S1). Ob-Rfl was demonstrated to be the main signal-transducing isoform [11] and Ob-R219.3 showed the highest shedding rate in our in-vitro experiments. Therefore, we focused further investigations in the present study on these two Ob-R-isoforms. ADAM10 predominantly mediates constitutive Ob-R shedding. The possible involvement of metalloproteases in constitutive Ob-R shedding was investigated by use of the broad spectrum metalloprotease inhibitor GM6001. The incubation of Ob-R219.3 transfected cells with GM6001 (25–100 mM) for 24 h and 48 h (Supporting information Fig. S2) decreased shedding by up to 52% (P,0.01). Metalloproteases ADAM10 and ADAM17 are probably the best characterized members of the ADAM family and they are known to catalyze shedding of membrane proteins which show structural similarities to Ob-R such as the interleukin 15 receptor alpha (IL-15Ra), the interleukin 6 receptor (IL-6R) and the growth hormone receptor [37–39]. To investigate the effect of ADAM10 and ADAM17 on Ob-R shedding, Ob-R219.3 transfected HEK cells were incubated with either one of two metalloprotease inhibitors: GI254023X (GI) that blocks ADAM10 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34787