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pmic7234-sup-0001-FigureS1.pdf286KFigure S1. A) Immunoblotting(right side) from the BN-PAGE (left side) revealed a single band at the approx. apparent MW of 480 kDa. B) Immunoblottingpatterns of the 2DE (BN/SDS-PAGE) from cellular membrane fraction and C) total cell lysateshow a single spot at the approx. MW of 75 kDacompatible with the 5-HT1AR-YPF. A rabbitpolyclonalantibody against mouse/rat 5-HT1AR was used.
pmic7234-sup-0002-FigureS2.pdf242KFigure S2. A) The BN Western blot pattern indicating the position for excision of bands in the right two lanes. B) The 3DE (BN/SDS/SDS-PAGE)immunoblottingof 5-HT1AR from the cellular membrane fraction showing a single spot C) from total celllysate.
pmic7234-sup-0003-FigureS3.pdf780KFigure S3. A) The gel and the correspondingimmunoblottingfrom proteinsimmunoprecipitatedby the GFP/YFP-antibody and the bracket indicates the presence of the tagged 5-HT1AR. Thecorrespondingnumbers indicate the bands excised for mass spectrometric analysis. B) Silver stained gel of different elution protocols. The best result was by SDS elution. C) The silver-stained 2DE gel of theimmunoprecipitatedproteins with an arrow indicating the 5-HT1AR. The corresponding numbers indicate the spots excised for massspectrometric analysis.
pmic7234-sup-0004-FigureS4.pdf52KFigure S4. Immunoblottingof 2DE IEF/SDS-PAGE of recombinant rat 5-HT1AR from transfectedtsA201 cells.
pmic7234-sup-0005-FigureS5.pdf710KFigure S5. Representative gel image of 1DE SDS-PAGE and 2DE dSDS-PAGE for mass spectrometricalanalysis.
pmic7234-sup-0006-FigureS6.pdf113KFigure S6. Sequence coverage, post-translationalmodifications and amino acid substitutions of recombinant rat 5-HT1AR obtained from bioinformatictools of Mascot v2.3 and Modiro™v1.1.
pmic7234-sup-0007-FigureS7.pdf1165KFigure S7. Sequence coverage and identified peptidesof YFP.
pmic7234-sup-0008-FigureS8.pdf114KFigure S8. MS/MS spectra of phosphorylatedand dephosphorylatedS301 by alkaline phosphatasetreatment from recombinant rat 5-HT1AR.
pmic7234-sup-0009-FigureS9.pdf44KFigure S9. Immunoblottinganalysis of recombinant rat 5-HT1AR from tsA201 cells after deglycosylatingenzyme treatment.
pmic7234-sup-0010-FigureS10.pdf1946KFigure S10. Representative mass spectra of identified post-translationalmodifications of recombinant rat 5-HT1AR expressed from tsA201 cells.
pmic7234-sup-0011-TableS1.doc37KTable S1. Sequence coverage of recombinant rat 5-HT1AR. Two different search engines (Mascot v2.3 and Modiro™ v1.1) were used. The conditions of all enzymes used in this study are listed. The sequence coverage was evaluated by combining sequence coverages of all spots of rat recombinant rat 5-HT1AR.
pmic7234-sup-0012-TableS2.xls235KTable S2. The list of identified peptides of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) with Mascot v2.3
pmic7234-sup-0013-TableS3.xls1066KTable S3. The list of identified peptides of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) with Modiro™ v1.11
pmic7234-sup-0014-S1.doc55KSupporting Information

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