Updated FTP location. TEC cells (K5D1 cTECs in quadruplicate, K5D1 mTECs in triplicate, and K5D1-β5tKO cTECs in triplicate) were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 8.0, and 2 mM DTT. The clarified lysates were reduced in 5 mM DTT and alkylated in 27.5 mM iodoacetamide. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate buffer. The proteins were digested with trypsin/Lys-C mix for 16 hr. Approximately 10 µg of peptides for each sample were labeled with 0.2 mg of TMT10-plex reagents for 1 hr at room temperature. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA and fractionated using Pierce high pH reversed-phase peptide fractionation kit. Ten fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% ACN. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resultant peptides (1 µg each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column. The mass spectrometer was operated in a data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3×106 and a mass range from 375 to 1,400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1×105, an isolation window of 0.4 m/z, a maximum injection time of 100 msec and a normalized collision energy of 32. Dynamic exclusion was set to 30 sec. Raw data were directly analyzed against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.2 with Mascot search engine version 2.5 for identification and TMT quantification. Peptides and proteins were filtered at a false-discovery rate (FDR) of 1 % using the percolator node and the protein FDR validator node, respectively.