As was the case for pFGFRs, we found HSPGs expressed in glial cells and AL neurons

o all institutional guidelines on the ethical use of animals in research. The protocols for the use of animals were approved by the Academia Sinica IACUC. All efforts were made to minimize pain and suffering. 8 Phosphorylation of Dcp1a by ERK Phosphorylation of Dcp1a by ERK Cell culture All cell lines were purchased from ATCC. HEK 293T and HeLa cells were cultured in DMEM supplemented with 3.7 g/l ATL 962 site sodium bicarbonate and 10% Gibco qualified FBS in a 5% CO2 humidified incubator at 37uC. 3T3-L1 preadipocytes were 9400011 cultured in DMEM supplemented with 1.5 g/l sodium bicarbonate and 10% bovine serum in a 5% CO2 humidified incubator at 37uC. Two days after reaching confluency, 3T3-L1 cells were stimulated to undergo differentiation into adipocytes by replacing fresh medium with an induction cocktail, FMDI, that was comprised of 10% FBS, 0.5 mM MIX, 5 mM DEX, and 1.7 mM bovine insulin. In the experiments with ERK kinase 23696131 inhibitors, 3T3-L1 cells were pretreated for 30 min with either 20 mM U0126 or the same volume of DMSO as a vehicle control 2 days after reaching confluency. Liquid chromatography -MS/MS analysis Nano-LCMS/MS experiments were performed on a LTQ-FT mass spectrometer equipped with a nanoelectrospray ion source in positive ion mode. Enzyme-digested cross-linked protein samples were injected onto a self-packed precolumn. Chromatographic separation was performed on a self-packed reversed phase C18 nanocolumn using 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile. A linear gradient from 5 to 40% mobile phase B for 40 min at a flow rate of 300 nL/ min was applied. A scan cycle was initiated with a full-scan survey MS spectrum performed on the FTICR mass spectrometer with resolution of 100,000 at 400 Da. The ten most abundant ions detected in this scan were subjected to an MS/MS experiment performed in the linear ion trap. Ion accumulation and maximal ion accumulation time for full-scan and MS/MS were set at 16106 ions, 1000 ms and 56104 ions, 200 ms, respectively. Ions were fragmented with collision-induced dissociation with a normalized collision energy of 35%, activation Q of 0.3, and activation time of 30 ms. For data analysis, all MS/MS spectra were converted to mzXML and mgf format from the experimental RAW file with MM File Conversion Tools, and then analyzed by MassMatrix for MS/MS ion search. The search parameters in MassMatrix including the error tolerance of precursor ions and the MS/MS fragment ions in spectra were 10 ppm and 0.6 Da, and the enzyme was assigned to be trypsin with the missed cleavage number of three. The variable post-translational modifications in the search parameters were assigned to include the oxidation of methionine and the phosphorylation of serine/threonine/tyrosine. Cell extract preparation Harvested cells were lysed with NET buffer containing protease and phosphatase inhibitor cocktails. For the CIP experiments, phosphatase inhibitor was not added to the NET buffer, and the cell lysates were treated with CIP at 37uC for 3 h. Co-IP assays HEK 293T cells were co-transfected with the indicated plasmids using the CaPO4 precipitation method. Cells were harvested in NET buffer 24 h after transfection and were centrifuged at 15,0006g for 10 min. The supernatants were immunoprecipitated using anti-Flag M2 agarose at 4uC for 2 h. After the immunoprecipitation mixture was washed three times with NET buffer, bound proteins were eluted by boiling in protein sample buffer, and western blot analys