SUPPLEMENTAL DATA Apratoxin a Reversibly Inhibits the Secretory
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Liu Y, Law BK, Luesch H (2009) Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. (manuscript number MOL 56085) SUPPLEMENTAL DATA Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation Yanxia Liu, Brian K. Law, and Hendrik Luesch Department of Medicinal Chemistry, University of Florida, 1600 SW Archer Road, Gainesville, Florida 32610, USA (Y.L., H.L.); and Department of Pharmacology & Therapeutics, University of Florida, 1376 Mowry Road, Gainesville, Florida 32610, USA (B.K.L.) 1 Liu Y, Law BK, Luesch H (2009) Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. (manuscript number MOL 56085) Supplementary Figure Supplementary Figure S1. Comparison of enzymatic deglycosylation (PNGase F), inhibition of glycosylation (tunicamycin) and apratoxin A treatment. Control U2OS cell lysates were deglycosylated with PNGase F (lane 1) and resulting PDGFR-β analyzed by immunoblot analysis and compared with lysates obtained from untreated U2OS cells and cells treated for 12 h with tunicamycin (500 ng/mL) or apratoxin A (50 nM). 2 Liu Y, Law BK, Luesch H (2009) Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. (manuscript number MOL 56085) Supplementary Table S1 Apratoxin A (50 nM) induced fold changes in levels of proteins enriched after cell surface a biotinylation of U2OS cells (quantified by iTRAQ labeling) Gene Symbol Annotation Peptidesb 1 h 4 h 12 h 24 h Receptors/cell surface proteins LRPAP1 low density lipoprotein receptor-related protein 8 –1.13 –1.61 –4.06 –3.79 associated protein 1 M6PR mannose-6-phosphate receptor (cation dependent) 4 +1.02 –1.07 –2.70 –1.54 CD151 CD151 molecule (Raph blood group) 1 –1.01 +1.02 –2.63 –1.37 NPTN neuroplastin 5 –1.51 –1.15 –2.37 –1.92 GPC1 glypican 1 7 +1.36 +1.02 –2.21 –1.65 ITGAV integrin, alpha V (vitronectin receptor, alpha 21 +1.15 –1.03 –2.04 –1.76 polypeptide, antigen CD51) NRD1 nardilysin (N-arginine dibasic convertase) 16 –1.16 +1.24 –1.87 –2.22 TFRC transferrin receptor (p90, CD71) 28 +1.01 –1.25 –1.83 –2.79 DAG1 dystroglycan 1 (dystrophin-associated glycoprotein 1) 13 –1.09 –1.25 –1.71 –2.26 ITGB1 integrin, beta 1 (fibronectin receptor, beta 20 –1.06 –1.06 –1.56 –1.98 polypeptide, antigen CD29 includes MDF2, MSK12) NCAM1 neural cell adhesion molecule 1 12 –1.03 –1.06 –1.51 –2.16 Other membrane proteins TMED2 transmembrane emp24 domain trafficking protein 2 3 –1.31 +1.08 –2.36 –2.25 TMED10 transmembrane emp24-like trafficking protein 10 4 +1.20 –1.16 –1.82 –2.78 (yeast) VAPB VAMP (vesicle-associated membrane protein)- 2 +1.04 –2.35 –1.77 –1.73 associated protein B and C CCDC47 coiled-coil domain containing 47 4 +1.07 +1.38 –1.35 –13.87 ANXA1 annexin A1 5 +1.61 –1.12 +2.01 +1.81 Protein disulfide isomerases PDIA3 protein disulfide isomerase family A, member 3 23 +1.10 +1.15 –1.65 –3.02 PDIA4 protein disulfide isomerase family A, member 4 17 –1.04 +1.17 –1.93 –4.15 PDIA6 protein disulfide isomerase family A, member 6 12 –1.15 –1.04 –1.71 –2.86 TXNDC4 thioredoxin domain containing 4 (endoplasmic 7 +1.10 –1.13 –1.78 –2.14 reticulum) TXNDC5 thioredoxin domain containing 5 8 +1.13 +1.31 –1.88 –3.20 P4HB procollagen-proline, 2-oxoglutarate 4-dioxygenase 23 –1.02 +1.16 –1.87 –3.80 (proline 4-hydroxylase), beta polypeptide Other ER proteins CALU calumenin 8 –1.19 +1.11 –2.52 –5.08 RCN1 reticulocalbin 1, EF-hand calcium binding domain 8 –1.08 +1.04 –2.39 –5.87 RCN2 reticulocalbin 2, EF-hand calcium binding domain 5 –1.14 –1.15 –2.13 –2.14 P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxygenase 11 –1.24 +1.01 –2.17 –2.68 (proline 4-hydroxylase), alpha polypeptide I PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 11 +1.05 –1.27 –1.69 –4.31 HSPA5 (BIP) heat shock 70kDa protein 5 (glucose-regulated 28 –1.02 +1.08 –2.08 –3.70 protein, 78kDa) PRKCSH protein kinase C substrate 80K-H (beta-subunit of 9 –1.11 +1.29 –2.08 –3.05 glucosidase II) LMAN1 lectin, mannose-binding, 1 11 –1.09 –1.13 –2.03 –2.67 HYOU1 hypoxia up-regulated 1 27 –1.14 +1.24 –2.02 –3.02 CANX calnexin 14 –1.08 –1.12 –2.02 –3.21 3 Liu Y, Law BK, Luesch H (2009) Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. (manuscript number MOL 56085) HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 38 ±1.00 +1.15 –1.95 –3.11 ERP29 endoplasmic reticulum protein 29 7 –1.03 +1.06 –1.80 –2.26 STT3A STT3, subunit of the oligosaccharyltransferase 8 +1.31 –1.11 –1.90 –1.99 complex, homolog A (S. cerevisiae) RPN1 ribophorin I 13 –1.06 –1.13 –1.77 –2.23 PPIB peptidylprolyl isomerase B (cyclophilin B) 14 –1.05 +1.36 –1.75 –3.32 CALR calreticulin 12 –1.15 +0.82 –1.69 –3.46 GANAB glucosidase, alpha; neutral AB 27 –1.06 +1.24 –1.64 –3.12 SERPINH1 serpin peptidase inhibitor, clade H (heat shock 15 +1.08 +1.09 –1.52 –2.00 protein 47), member 1, (collagen binding protein 1) UGCGL1 UDP-glucose ceramide glucosyltransferase-like 1 17 –1.28 –1.08 –1.06 –2.77 tRNA synthetases & protein biosynthesis RARS arginyl-tRNA synthetase 13 +1.04 –1.02 +1.43 +2.21 QARS glutaminyl-tRNA synthetase 15 –1.20 –1.15 +1.44 +1.47 DARS aspartyl-tRNA synthetase 9 –1.06 –1.15 +1.56 +1.85 KARS lysyl-tRNA synthetase 15 +1.06 +1.07 +1.59 +1.41 TARS threonyl-tRNA synthetase 11 –1.35 +1.13 +1.86 +1.29 IARS isoleucyl-tRNA synthetase 21 +1.63 –1.04 +2.02 +1.69 RPL12 ribosomal protein L12 3 +1.27 +1.66 +2.32 +2.15 EIF3D eukaryotic translation initiation factor 3, subunit D 3 –1.61 –1.43 +2.39 +1.11 SRP72 signal recognition particle 72kDa 12 +1.32 –1.08 +2.55 +1.50 Mitochondrial proteins GPD2 glycerol-3-phosphate dehydrogenase 2 10 +1.37 +1.08 –1.61 –2.55 (mitochondrial) HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 6 –1.25 –1.20 –1.47 –2.15 LOC647153 similar to voltage-dependent anion channel 2 6 –1.01 –1.02 –1.23 –2.06 Cytoskeletal proteins TPM1 tropomyosin 1 (alpha) 12 –3.65 –1.15 –1.40 –1.62 MYL6 myosin, light chain 6, alkali, smooth muscle and non- 5 –1.29 –1.14 +1.13 +2.44 muscle MYH9 myosin, heavy chain 9, non-muscle 53 +1.19 –1.19 +1.17 +3.15 Other proteins PSMA4 proteasome (prosome, macropain) subunit, alpha 6 –1.63 –1.04 –2.64 –4.00 type, 4 MESDC2 mesoderm development candidate 2 3 +2.71 +1.09 –2.13 –4.19 TBRG4 transforming growth factor beta regulator 4 8 –1.06 +1.22 –2.08 –1.42 PRDX4 peroxiredoxin 4 8 +1.01 +1.05 –1.84 –2.45 RAB1B RAB1B, member RAS oncogene family 9 –1.38 –1.02 –1.49 –2.26 RAB2A RAB2A, member RAS oncogene family 4 –1.40 +1.20 –1.09 –3.33 GNAI2 guanine nucleotide binding protein (G protein), alpha 11 +1.16 +1.08 –1.31 –2.00 inhibiting activity polypeptide 2 SCD stearoyl-CoA desaturase (delta-9-desaturase) 3 –1.22 –1.03 –1.29 –2.38 CDC42 cell division cycle 42 (GTP binding protein, 25kDa) 5 +1.08 –1.15 –1.22 –2.80 C14orf166 chromosome 14 open reading frame 166 5 +1.49 +2.10 +1.24 –1.16 PA2G4 proliferation-associated 2G4, 38kDa 6 +1.29 –2.31 –1.25 –1.31 PGAM1 phosphoglycerate mutase 1 (brain) 4 –1.17 –1.29 +1.39 +2.11 ALDOA aldolase A, fructose-bisphosphate 16 +1.10 –1.08 +1.53 +2.21 PRMT1 protein arginine methyltransferase 1 4 +1.14 +1.24 +2.09 –1.01 a 95% confidence level. b Used for protein identification. 4 .