Supplementary Materials Supplemental Figures and Tables 155825_1_supp_433971_q1hkyb. soluble proteins. The pellet was dissolved in a altered RIPA buffer (50 mm Hepes, pH 7.5, 150 mm Cevimeline hydrochloride hemihydrate NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1% SDS, and protease inhibitors), and the supernatant after a centrifugation was designated as membrane proteins. Equal amounts of soluble proteins and equal amounts of membrane proteins from the two cell populations were mixed, respectively, and fractionated by a 12% SDS-PAGE gel (Bio-Rad Mini gel; 7.2 cm x 8.6 cm) for LC-MS/MS analysis. LC-MS/MS and Data Analysis In-gel digestion, database search, and quantification of LC-MS/MS data were performed as described previously (28, 29). Specifically, the entire lane of the Coomassie brilliant blue-stained gel was cut into 10 slices, followed by in-gel digestion with trypsin (Promega, Madison, WI) (30, 31). The resulting peptides were analyzed by LC-MS/MS using a LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, Waltham, MA) operated in a data-dependent mode for tandem MS as described previously (28). Natural data from LC-MS/MS analysis were processed by MaxQuant (version 1.6.2.10) (32, 33) with the built-in search engine Andromeda (34) and searched against a target-decoy (35) human SwissProt protein database (November 2018; 20,408 entries) retrieved from UniProt (www.uniprot.org). The false discovery rates (FDRs) for peptide and protein identification were both set Rabbit Polyclonal to CDC25A (phospho-Ser82) to 1%. The MS error tolerance was set to 4.5 ppm, and the MS/MS error tolerance was set to 20 ppm. The minimum required peptide length was set to 7 amino acids, and a maximum of 2 missed cleavages was allowed. The variable modifications of acetylation at peptide N terminus and oxidation on methionine, and the fixed modification of cysteine carbamidomethylation were included. SILAC ratios (radioresistant/parental protein expression ratios; light/heavy ratios) were calculated using unique and razor peptides with a minimum ratio count of 2 Cevimeline hydrochloride hemihydrate (33). The proteins that matched to the reverse database, identified only by site, and common contaminants were removed. The proteins that were identified by single peptide were also discarded. The remaining proteins were analyzed by Perseus (version 1.6.2.2) (36), and the Significance B score was obtained for the quantified proteins. The Significance B score is usually a significance score for protein SILAC ratios and identifies outliers based on the standard deviation of the protein SILAC ratios of the main distribution and signal intensity (32). A protein was a differentially expressed protein if (1) Cevimeline hydrochloride hemihydrate its ratio was significant by the Significance B score with 0.05, and (2) a log2 fold change was larger than 1.5 (representing an actual fold change of 2.82). The significantly changed soluble proteins and membrane proteins were combined and analyzed by the Core Analysis module of the software IPA (Ingenuity Pathway Analysis; Ingenuity? Systems, Redwood City, CA), a bioinformatics tool based on information from published literature (37). For the proteins that were identified in both soluble and membrane fractions (the shared proteins), an average of the soluble and membrane SILAC ratios was used for each protein in the IPA analysis (see Results). Quantitative Real-Time PCR (qRT-PCR) qRT-PCR was performed as described previously Cevimeline hydrochloride hemihydrate (28, 38). Briefly, total RNA was isolated using RNeasy Mini Kit according to manufacturer’s instructions (Qiagen, Valencia, CA). One g of RNA was reverse transcribed using an iScript cDNA Synthesis Kit following the manufacturer’s protocol (Bio-Rad, Hercules, CA), and the resulting cDNA was used for qRT-PCR analysis of the human genes NT5E (protein, CD73), EGFR, OSM, PDGFB, TGFB1 (TGF-1), TGFB2 (TGF-2), TGFB3 (TGF-3), Cevimeline hydrochloride hemihydrate FN1, CDH1 (E-cadherin), ZEB1, and PLAU (urokinase-type plasminogen activator). mRNA abundance was measured using SYBR Green Supermix (ThermoFisher Scientific) from three impartial sample preparations. Relative gene expression was calculated according to the traditional 2?Ct method (39). The primer sequences used for qRT-PCR analysis in this study were listed in supplemental Table S8. Western Blotting Western blotting was performed as described previously (40). Specifically, cell lysates were extracted in a altered RIPA buffer (50 mm Hepes, pH7.5, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% Glycerol, 1% Triton X-100, 1% SDS and protease inhibitors; for detection of BAD phosphorylation, 1 mm sodium orthovanadate, 10 mm sodium fluoride, and 10 mm -glycerophosphate were included as phosphatase inhibitors). The proteins were separated on a SDS-PAGE (60 g protein/lane), transferred to a nitrocellulose membrane (Millipore, Billerica,.