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SUPPLEMENTAL RESEARCH DESIGN AND METHODS
Details of PPI-EGFPs construcs
We made four different PPI-EGFP constructs as described below. (A) PPI-EGFP. PPI-EGFP cDNA was constructed using a strategy similar to that of Ohara-Imaizumi et al ADDIN REFMGR.CITE Ohara-Imaizumi2004OI2004TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cellsJournalOI2004TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cellsOhara-Imaizumi,M.Nishiwaki,C.Kikuta,T.Nagai,S.Nakamichi,Y.Nagamatsu,S.2004/7/1ImmunohistochemistryNot in File1318Biochem.J.381Pt 1Department of Biochemistry (II), Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, JapanPM:15128287Biochem.J.1(13). Briefly, human PPI cDNA pchi119 (provided by Professor Graeme I. Bell, Howard Hughes Medical Institute, The University of Chicago, Chicago, USA), lacking a TGA stop codon, was amplified by PCR using forward primer, 5-GAATTCCGGGGGTCCTTCTGCCATG-3 (primer #1; EcoRI site underlined), and reverse primer, 5-GGATCCCAGTTGCAGTAGTTCTCCAGC-3 (primer #2; BamHI site underlined), where TGA was replaced by TGG. The PCR product was subcloned into pCR2.1 vector (Invitrogen). The PPI cDNA fragment lacking a stop codon was cleaved with EcoRI/BamHI and subcloned into the EcoRI/BamHI site of the multiple cloning site of pEGFP-N1 (Clontech, see supplemental Fig. 1A). (B) Kozak-PPI-EGFP. To generate a construct which contained a Kozak sequence, human PPI cDNA was amplified using forward primer 5-GAATTCGTCGCCACCATGGCCCTGT-3 (primer #3; EcoRI site underlined) and primer #2. The resulting product was cleaved and subcloned into pEGFP-N1 vector (supplemental Fig. 1B). (C) 0Trp-PPI-EGFP. To generate a construct which lacked TGG (tryptophan, Trp) sequence, human PPI cDNA was amplified using primer #1 as forward primer and reverse primer 5-GGATCCTTGCAGTAGTTCTCCAGCTGGTA-3 (primer #4; BamHI site underlined). The resulting product was cleaved and subcloned into pEGFP-N1 vector (supplemental Fig. 1C). (D) 2Trp-PPI-EGFP. A construct containing a TGG TGG (Trp Trp) sequence was generated by amplifying human PPI cDNA using primer #1 as forward primer and reverse primer 5-GGATCCCACCAGTTGCAGTAGTTCTCCA-3 (primer #5; BamHI site underlined). The resulting product was cleaved and subcloned into pEGFP-N1 vector (supplemental Fig. 1D). The sequence of all plasmid inserts was verified by automated sequencing.
Western immunoblotting
Three days after transfection, MIN6 cells (one confluent 25 cm2 flask) expressing PPI-EGFP, Kozak-PPI-EGFP, 0Trp-PPI-EGFP, 2Trp-PPI-EGFP or EGFP alone, as a control, were harvested and washed twice in ice-cold PBS, scraped into ice-cold lysis buffer (1% Triton X-100, 5 g/ml pepstatin, 5 g/ml antipain, 5 g/ml, leupeptin, 2 mmol/l benzamidine, 0.5 mmol/l DTT in PBS). After solubilization with 1% Triton X-100, insoluble material was removed by centrifugation. Total protein extracts (50 g) were resolved by 10% SDS-PAGE and transferred to nitro-cellulose membranes, followed by immunoblotting with mouse monoclonal anti-GFP antibody (Roche Diagnostics, 1:1000 dilution). HRP-conjugated goat anti-mouse IgG (Sigma, 1:5000 dilution) was revealed by using a BM chemiluminescence blotting substrate (Roche Diagnostics).
Immunocytochemistry
Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 as described in detail elsewhere ADDIN REFMGR.CITE Pouli1998P1998ASecretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaeraJournalP1998ASecretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaeraPouli,A.E.Emmanouilidou,E.Zhao,C.Wasmeier,C.Hutton,J.C.Rutter,G.A.1998/7/1Adrenal Gland NeoplasmsanalysisAnimalsbiosynthesisChimeric ProteinsComparative StudyCytoplasmic GranulesExocytosisgeneticsImmunohistochemistryInsulinomaIslets of LangerhansLuminescent ProteinsMembrane GlycoproteinsMembrane ProteinsmetabolismMicroscopy,ConfocalNeoplasm ProteinsPancreatic NeoplasmspathologyPheochromocytomaphysiologyProtein-Tyrosine-PhosphataseRatsSupport,Non-U.S.Gov'tTumor Cells,CulturedultrastructureNot in File193199Biochem.J.333 (Pt 1)Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, U.KPM:9639579Biochem.J.1Tsuboi2003T20035'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamicsJournalT20035'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamicsTsuboi,T.daSilvaXavier,GLeclerc,I.Rutter,G.A.2003/12/26ActinsAdenosine TriphosphateAdenoviridaeAnimalsBinding SitesCalciumCell MembraneDigitoninDose-Response Relationship,DruggeneticsGlucoseHydrogen-Ion ConcentrationImmunohistochemistryInsulinIslets of LangerhansKineticsLuminescent ProteinsMagnesiummetabolismMiceMicroscopy,ConfocalModels,StatisticalMultienzyme ComplexesPhosphorylationphysiologyPlasmidsProtein-Serine-Threonine KinasesSecretory VesiclesSupport,Non-U.S.Gov'tTime FactorsTubulinNot in File5204252051J.Biol.Chem.27852Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United KingdomPM:14532293J.Biol.Chem.1Varadi2002V2002Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cellsJournalV2002Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cellsVaradi,A.Ainscow,E.K.Allan,V.J.Rutter,G.A.2002/11/1Adenosine TriphosphateAnimalsCells,CulturedClone CellsCytosoldiagnostic useDose-Response Relationship,Drugdrug effectsExocytosisgeneticsGlucoseGreen Fluorescent ProteinsHumansInsulinIslets of LangerhansKinesinLuminescent ProteinsMembrane ProteinsmetabolismMitochondriaMutationpharmacologyProtein TransportProtein-Tyrosine-PhosphataseProteinsRecombinant Fusion ProteinsResearch Support,Non-U.S.Gov'tsecretionSecretory VesiclesNot in File41774189J.Cell Sci.115Pt 21Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UKPM:12356920J.Cell Sci.1(10;11;16). Cells were labeled with a rabbit polyclonal anti-phogrin antibody which cross-reacts with the cytosolic C-terminal domain of phogrin (1:200 dilution), then processed with Alexa 568 conjugated anti-rabbit IgG antibody (Molecular Probes, 1:3000 dilution). Immunofluorescence staining was observed on a Leica TCS-AOBS laser-scanning confocal microscope.
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Linear maps of constructs. (A) PPI-EGFP; (B) Kozak-PPI-EGFP; (C) 0Trp-PPI-EGFP; (D) 2Trp-PPI-EGFP. All inserts were subcloned into vector pEGFP-N1 under the control of the CMV immediate early gene promoter.
Supplemental Figure 2. Localization of PPI-EGFP constructs within MIN6 cells. (A) Confocal image of paraformaldehyde-fixed MIN6 cells showing the distribution of PPI-EGFP, (B) Kozak-PPI-EGFP, (C) 0Trp-PPI-EGFP and (D) 2Trp-PPI-EGFP. This vesicular pattern was observed in about 60% of cells (See Supplemental Table1). (E) cytosol and nucleus fluorescence of PPI-EGFP (observed about 10% of cells) and (F) minor vesicular fluorescence of PPI-EGFP (observed about 30% of cells). (G) Confocal image of paraformaldehyde-fixed PPI-EGFP fluorescence. (H) Image of Alexa 568-labeled phogrin fluorescence in the same cell. (I) Overlay of (G) and (H). The scale bar represents 5 mm . T h u s , t h e s e d a t a i n d i c a t e t h a t a p u n c t u a t e d i s t r i b u t i o n o f v e s i c l e s r e f l e c t e d t a r g e t i n g o f c o n s t r u c t t o d e n s e c o r e v e s i c l e s . ( J ) P P I a n d E G F P r e m a i n e d t o g e t h e r a s a s i n g l e p r o t e i n . M I N 6 c e l l s t r a n s f e c t e d w i t h t h e P P I - E G F P ( l a n e 2 ) , K o z a k - E G F P ( l a n e 3 ) , 0Trp-PPI-EGFP (lane 4), 2Trp-PPI-EGFP (lane 5) and EGFP (lane 1) as control. Therefore, these results demonstrating that mistargeting was not due to proteolytic cleavage and liberation of free EGFP. Cell lysates were analyzed by 10% SDS-PAGE and immunoblotting with mouse monoclonal anti-GFP antibody and HRP-conjugated goat anti-mouse IgG. The position of the molecular mass marker (X10-3) is shown on the left.
Supplemental Table 1
Vesicular targeting efficiency of Phogrin-EGFP, NPY-Venus and PPI-EGFP constructs
Constructs MIN6 INS-1 Islets (rat)
Phogrin-EGFP 82.1 2.2 % (179 cells, n = 9) 82.4 2.9 % (188 cells, n = 9) 89.1 2.4 % (49 islets, n = 5)
NPY-Venus 80.4 2.1 % (144 cells, n = 9) 81.3 1.8 % (150 cells, n = 9) 85.4 3.9 % (40 islets, n = 5)
PPI-EGFP 56.4 7.2 % (167 cells, n = 9)*** 51.4 4.7 % (168 cells, n = 9)*** 65.1 4.1 % (49 islets, n = 5)**
Kozak-PPI-EGFP 47.1 5.1 % (190 cells, n = 9)*** 39.9 4.6 % (170 cells, n = 9)*** 62.3 ) H 9 : i o ̽}}}n^ h,\ CJ OJ PJ QJ ^J aJ h,\ >*CJ OJ QJ ^J aJ #j h,\ CJ OJ QJ U^J aJ h,\ CJ OJ QJ ^J aJ o(h,\ CJ OJ QJ ^J aJ h,\ 6CJ OJ QJ ^J aJ o( h,\ 6CJ OJ QJ ^J aJ h,\ CJ OJ QJ \^J aJ o( #h,\ 56CJ OJ QJ ^J aJ o( h,\ 5CJ OJ QJ ^J aJ o(# ) H 5 6 M 3 3 4 4 4 ; ; ; <