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 (13). Briefly, human PPI
cDNA pchi1–19 (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-PPIEGFP. A construct containing a TGG TGG (Trp Trp) sequence was generated by amplifying
1
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 µg/ml
pepstatin, 5 µg/ml antipain, 5 µg/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 µg) 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
2
Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 as described in detail
elsewhere (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.
3
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-PPIEGFP, (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 m. Thus, these data indicate that a punctuate distribution of vesicles reflected
targeting of construct to dense core vesicles. (J) PPI and EGFP remained together as a single
protein. MIN6 cells transfected with the PPI-EGFP (lane 2), Kozak-EGFP (lane 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 antiGFP antibody and HRP-conjugated goat anti-mouse IgG. The position of the molecular mass
marker (X10-3) is shown on the left.
4
Supplemental Table 1
Vesicular targeting efficiency of Phogrin-EGFP, NPY-Venus and
PPI-EGFP constructs
Constructs
MIN6
Islets (rat)
Phogrin-EGFP
82.1 ± 2.2 % (179 cells, n = 9)
= 9) 89.1 ± 2.4 % (49 islets, n = 5)
NPY-Venus
80.4 ± 2.1 % (144 cells, n = 9)
= 9) 85.4 ± 3.9 % (40 islets, n = 5)
PPI-EGFP
56.4 ± 7.2 % (167 cells, n = 9)***
= 9)*** 65.1 ± 4.1 % (49 islets, n = 5)**
Kozak-PPI-EGFP 47.1 ± 5.1 % (190 cells, n = 9)***
= 9)*** 62.3 ± 4.1 % (21 islets, n = 5)**
0Trp-PPI-EGFP
44.3 ± 6.9 % (159 cells, n = 9)***
= 9)*** 64.5 ± 4.9 % (22 islets, n = 5)**
2Trp-PPI-EGFP
49.1 ± 3.5 % (145 cells, n = 9)***
= 9)*** 61.3 ± 2.5 % (25 islets, n = 5)**
INS-1
-
82.4 ± 2.9 % (188 cells, n
81.3 ± 1.8 % (150 cells, n
51.4 ± 4.7 % (168 cells, n
39.9 ± 4.6 % (170 cells, n
35.4 ± 3.4 % (132 cells, n
45.8 ± 3.1 % (136 cells, n
** p< 0.01 and *** p<0.001, when compared with Phogrin-EGFP or NPY-Venus
5