www.sciencemag.org/cgi/content/full/325/5945/1250/DC1

Supporting Online Material for

Rab35 Controls Actin Bundling by Recruiting Fascin as an Effector Protein

Jun Zhang, Marko Fonovic, Kaye Suyama, Matthew Bogyo, Matthew P. Scott*

*To whom correspondence should be addressed. E-mail: mscott@stanford.edu

Published 4 September 2009, Science 325, 1250 (2009) DOI: 10.1126/science.1174921

This PDF file includes: Materials and Methods

Figs. S1 to S12

Table S1

References

Supporting Online Material

MATERIALS AND METHODS

DNA constructs and fly strains

Rab35 and fascin cDNAs were amplified from Drosophila embryo total RNA, mouse

embryo total RNA, or HeLa cells. Site-directed mutagenesis (Stratagene) was performed to

generate the Rab35S22N, fascinS39A or fascinS39D. N-terminal YFP-,

myc-, FLAG-, tdTomato-tagged or non-tagged pUAST and pCDNA3.1 construct fused with

the Gateway cassette fragment (Invitrogen) were used to generate the different finalversions

of Rab35 and the fascin transgene, as in our previous report (S1). pWIZ-Rab35RNAi was

generated by inserting full length tail–tail inverted repeats of

Rab35 full-length cDNA into pWIZ (white Intron Zipper) vector, a previously described

RNAi construct useful for making transgenic flies (S2). A full length 5’-3’ Rab35 cDNA

sequence (about 600bp) was generated and inserted into NotI and SpeI restriction enzyme

sites located in the left arm of mini-white intron 2 in the pWIZ construct. A full-length

3’-5’ Rab35 cDNA sequence was inserted into the XbaI site in the the right arm of miniwhite

intron 2. Total RNA was isolated from embryos from control flies (yw) and flies expressing

Rab35RNAi and subjected to RT-PCR with as many as 40 amplification cycles. Rab1, Rab2,

and actin mRNA levels were shown as controls.

Plasmids encoding mitochondria-targeted Rab35 (Rab35-mito) proteins were constructed

using the different Rab35 cDNA versions (WT or DN) fused to cDNA fragments

corresponding to the hydrophobic C-terminal region

(LILAMLAIGVFSLGAFIKIIQLRKNN) of Listeria monocytogenes ActA protein (S3).

pG-SUPER-Fascin-Th, which expresses small hairpin RNA (shRNA), was obtained from

Dr. Danijela Vignjevic and was generated originally by Dr. Reuven Agami and colleagues

(S4).

Other fly stocks include tubulin-gal4 (a gift from Dr. Liqun Luo), prospero-gal4 (a gift from

Dr. Bruce Baker), CY2-gal4 (a gift from Dr. Trudi Schupbach), and the fascin mutant flies

sn1 (from the Bloomington Drosophila Stock Center).

Purified DNA containing each construct was injected to establish transgenic

Drosophila lines. Multiple lines of flies that carried each P element were recovered and

analyzed. All fly crosses described in this study were performed on standard media at 25°C

unless otherwise specified.

Rab35 antibody generation

Plasmids coding for full-length or the C-terminal 43 amino acids of Drosophila

Rab35 (NMFLSITRQVLDHKLRTSPNEQQKDTLHLKPNPKGSKGGKCCR) were

subcloned into the pATH10 vector to generate a transient receptor potential E (TrpE) fusion

protein for rabbit immunization. The same fragments were cloned into the pGEXKG vector

to produce glutathione S-transferase-tagged proteins, which were used to affinity-purify the

resulting antiserum. The antisera were purified using the AminoLink

Plus Immobilization Kit from Pierce. Elution of the antibody from the column was achieved

using 100mM glycine. Antibody specificity was tested (Fig. S12).

Cell Culture, immunostaining and imaging

Drosophila S2 cells (a line derived from embryos) and mammalian HeLa and

NIH3T3 cells were cultured following standard procedures. 2 x105 cells were seeded in a

24-well plate one day prior to transfection. Cells were transfected using Effectene

(Qiagen). 200 ng DNA was used in total for each well. Cells were fixed in 4%

paraformaldehyde for 15 min at room temperature or by methanol for 10 mins at 4°C 24-

72 hours after transfection, depending on the experimental conditions. Primary antibodies

against Rab35 and fascin (Santa Cruz Biotechnology) were used at a dilution of 1:100.

Secondary antibodies conjugated to Alexa Fluor 488, 594 (Molecular Probes) were used at a

dilution of 1:1000. All antibody incubations were performed at 4°C overnight in the presence

of 1% BSA. Other reagents used include Texas Red-phalloidin

(Molecular probes), phalloidin-488 (Molecular probes), mitotracker 580 (Molecular probes),

latrunculinA (EMD Biosciences), and nocodazole (Sigma). Latrunculin A (50

nM) or nocodazole (30 µM) were administered to the cells for 30 min. All fluorescent images

were taken on a Leica TCS-SP5 confocal microscope.

Affinity Chromatography, SDS-PAGE and silver stain

GST-Rab35 affinity chromatography with Bovine brain cytosol was performed as described

(S5). Bovine brain cytosol (obtained from Rancho Feeding Corporation,

Petaluma, CA) proteins were prepared and incubated with glutathione-sepharose beads

complexed with GST-Rab35-GTPγS or GST-Rab35-GDP. GTPγS and GDP were purchased

from Sigma. Eluted proteins were separated using SDS-PAGE and stained with a mass

spectrometry-compatible silver stain. Gels were fixed in a 45% methanol and 5% acetic acid

solution for two hours and rinsed in a 50% methanol solution for 20 minutes. After rinsing

twice with distilled water for 15 minutes, the gels were sensitized with 0.02% Na2S2O3 for

10 minutes, followed by staining with silver nitrate solution

(0.75g/500ml) for 20 minutes. Gels were developed with 0.04% formaldehyde and 2%

Na2CO3 solution.

Protein identification by mass spectrometry

Silver-stained protein bands were cut from the gel and destained as previously described

(S6). Protein bands were reduced in 25mM ammonium bicarbonate containing

10mM DTT (45 min at 56°C) and alkylated in the same buffer containing 55 mM

iodoacetamide (30 min at room temperature). Gel pieces were washed with 25 mM

ammonium bicarbonate and dried. We added trypsin solution (12.5 ng/µl trypsin in 25

mM NH4HCO3), and the samples were incubated overnight at 37°C. Digested peptides were

extracted from the gel with 50%ACN 5% formic acid. Extracts were concentrated to 10 µl

and analyzed by mass spectrometry.

Samples were analyzed on a LCQ DecaXP Plus ion trap mass spectrometer

(Thermo Fisher) coupled to a nanoLC liquid chromatography unit (Eksigent). Peptides were

separated on a BioBasic Picofrit C18 capillary column (New Objective). Elution was

performed with acetonitrile gradient from 0-50% in the 0.1% solution of formic acid over 40

min with overall flowrate of 350 nL/min. The three most intense base peaks in each scan

were analyzed by MS/MS. Dynamic exclusion was set at a repeat count of 2 with exclusion

duration of 2 min. Database searches were performed using the NCBInr protein database

using the Sequest algorithm (Thermo Scientific). Peptides with XCorr values over 1.5 (+1

charge), 2 (+2 charge) and 2.5 (+3 charge) and ΔCn values over 0.1 were considered for

further evaluation. Protein and peptide hits were statistically reevaluated by Scaffold

(Proteome Software). Peptide identifications were accepted if they could be established at

greater than 95% probability as specified by the Peptide Prophet algorithm (S7). Protein

identifications were accepted if they could be established at greater than 99.0% probability

and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein

Prophet algorithm (S8).

Biochemical procedures

GST-Rab35, GST-Rab5, GST-Rab2 and GST-fascin fusion proteins were expressed and

purified as described previously (S9). GST-fascin was cleaved with thrombin (7.5 U of

enzyme [GE healthcare] per mg of protein, at 4°C overnight) to remove the GST-tag. Rab

fusion proteins (1-2 µg) were incubated with fascin and glutathione-sepharose beads in GST

binding buffer for 3 h at 4°C, washed with PBS with 0.1% NP40 and 0.1% Triton X-100, and

subjected to SDS-PAGE. Immuno blot analysis was performed using anti-GST antibody

(1:5000, Abcam) as primary antibody and Donkey anti-goat HRP (1:2000, Jackson lab) as

secondary antibody. In vitro actin-bundling by Rab35 and fascin was measured using a

sedimentationbased assay with an actin binding spin-down assay kit, following the

manufacturer's protocol (Cytoskeleton Inc.). In brief, a low speed centrifugation was

performed to detect F-actin bundling activity. Each protein samples were incubated with or

without non-muscle F-actin for 30-40 mins at room temperature and the subjected to a

centrifugation at 14 000 g for 1 h at 24°C. Both supernatant and pellets were collected and

separated using SDS-PAGE. Gels were then stained with 0.1% of Coomassie blue to show

protein levels in each fraction.

Co-immunoprecipitation (coIP) was performed using HeLa cell extracts containing 1-2 mg of

protein and anti-myc (Santa Cruz Biotechnology). Precipitations were overnight at 4°C in a

standard RIPA buffer containing 1mM MgCl and GTPγS or GDP, followed by incubation

with protein G beads for 3 h at 4°C. Immuno blot analysis was performed using anti-flag

antibody (1:1000, Sigma), or anti-Drosophila fascin (singed) antibody (1:50, Developmental

studies Hybridoma Bank) as primary antibody and sheep anti-mouse HRP (1:2000, Jackson

lab) as secondary antibody.

Membrane preparation and cell fractionation

Procedures for fractionation were adapted from a previous study (S10). HeLa cells were

plated and transfected with Rab35 and fascin. For each condition, cells from four 150x15mm

culture dishes were washed once with PBS and scraped into a total of 4 ml of buffer (0.25 M

sucrose, 1 mM EDTA, 10 mM Hepes-NaOH, 1mM MgCl, pH 7.4) containing GTPγS or

GDP and complete protease inhibitor cocktail (Roche). Cells were broken by 10 passes

through a 27 gauge needle and centrifuged at 1000 g for 10 min at 4 °C. The post-nuclear

supernatant was centrifuged using an SW-55 swinging bucket rotor (Beckman Coulter) for

60min at 100 000 g at 4 °C. Pellets were resuspended in 300ul of 30% (w/v) OptiPrep (Axis

Shield) and loaded at the bottom of a step density gradient of iodixanol (2.5%, 10%, 17.5%

and 25%). The gradient was centrifuged for 3 h at 100 000 g at 4 °C. Eight fractions were

collected from each gradient. Samples were separated using SDS-PAGE and blotted with

primary antibodies against myc (1:1000), flag (1:1000), Sodium-Potassium ATPase (detects

plasma membrane; 1:2000, Abcam), calnexin (detects endoplasmic reticulum; 1:1000,

Abcam), and prohibitin (detects mitochondria; 1:1000, Abcam).

Data quantification and statistics

All quantification analysis was performed using the program Image J (from the National

Institutes of Health, http://rsb.info.nih.gov/ij/). To quantitate the intensity of signals on a

immunoblot, the digitized data were converted to 8-bit grayscale images. The signal areas

were selected with the “freehand selections” tool and both the Mean value (the average of the

gray intensity in selected area) and the Pixels value (the number of pixels contained in

selected area) were recorded for final comparison. To quantitate numbers of filopodia/cell

protrusions, cells were imaged using a Leica TCS-SP5 confocal microscope and a 63X oil-

immersion objective. Images of filopodia were overexposed to allow viewing details. Counts

of filopodia (cell protrusions) for 10 cells from three independent repeats of each treatment

were performed. The mean and standard deviation were calculated by Student’s t-test to

compare statistical significance of test materials against the control. Results of P<0.05 were

considered significant (differences P<0.05 denoted by * and P<0.01 denoted by **). Values

are shown as means ±SD.

Supplementary Figure 1. Reducing Rab35 levels in developing bristles using RNAi

causes defects similar to expressing Rab35DN.

A. Strategy for making the UAS-Rab35RNAi construct (Materials and Methods).

B. Flies with ubiquitous expression of Rab35RNAi, directed by tubulin-gal4, have no Rab35

mRNA detectable by RT-PCR even with 40 amplification cycles. Transcript levels for other

Rab genes, such as Rab1 and Rab2, were not affected. The actin mRNA level was shown as

internal control.

C-H. In situ hybridization (F-H, stages 9, 13, and 15, respectively) and

immunohistochemical detection of proteins levels (I-K, stages 13, 15, and 15, respectively)

showed that Rab35 transcripts are ubiquitous and somewhat enriched in the embryonic

nervous system. F-J, lateral views of fly embryos; K, dorsal view of fly embryos. Scale bar,

50 µm.

Supplementary Figure 2. Developing bristles of Rab35DN producing flies.

Rab35DN-producing flies showed a wavy, loose, thin, and disconnected actin structure (B)

compared to controls (A) at 45-47 h APF. Actin staining of Rab35DN bristles tapered off to

the tip (circled in B) compared to controls (circled in A). Texas Red-phalloidin staining

detected the packed actin bundles. Bars, 10 µm.

Supplementary Figure 3. Rab35 induces morphology changes in cultured cells.

Here we provide additional examples of cells treated and stained as in Figures 1G and

1K. YFP-Rab35WT (A-D), but not Rab35DN (E-H), induced filopodia-like membrane

protrusions in Drosophila S2 cultured cells. Green, YFP proteins; red, Phalloidin (F-actin).

Scale bars, 5 µm.

J. Quantification of filopodia numbers induced by Rab35WT. Rab35WT induced

42.9±7.83 filopodia compared to cells producing YFP alone (11.5±3.92, p<0.01).

Rab35DN blocked filopodia formation (2.0±1.69), compared to the control of YFP alone

(p<0.01).

Supplementary Figure 4. Over-production of mouse Rab35 has the same effect as over-

production of Drosophila Rab35 in developing bristles and cultured cells.

A. Sequence similarity of mouse and Drosophila Rab35.

B-C. Flies producing mouse Rab35DN displayed kinked bristles (arrows in C), compared to

controls (arrows in B). Scale bar, 0.2 mm.

D-F. YFP-mouse Rab35WT induced massive filopodia-like protrusions (E) in NIH3T3 cells,

which YFP alone did not (D). The induction of filopodia was not observed in YFP-mouse

Rab35DN-producing cells (F). Scale bars, 5 µm.

Supplementary Figure 5. Active Rab35 associates with fascin.

A. Bovine brain cytosol proteins that bound to GST-Rab35-GTPγS or GST-Rab35-

GDP were separated using SDS-PAGE. Candidate protein bands were cut out and

subjected to mass spectrometry (Material and Methods).

B. Drosophila S2 cell lysates were subjected to immunoprecipitation with anti-Rab35

antibody, followed by immunoblotting with anti-fly fascin monoclonal antibody to detect

fascin (top panel). Lower panel, fly Rab35. Cell lysates were pre-incubated with either

GTPγS or GDP to lock Rab35 in either its active or inactive form. Stronger binding of

fascin to Rab35+GTPγS (lane 3) than to Rab35+GDP (lane 4) (4.19±0.53 fold, n = 3,

p<0.05) was observed.

C. GST pull-down of Rab35 proteins that interact with fascin. Purified fascin was incubated

with beads loaded with GST-Rab35, in the presence of either GTPγS or GDP. A significantly

higher amount of fascin bound to Rab35+GTPγS (lane 1) than to Rab35+GDP (lane 2)

(3.33±0.64 fold, n = 3, p<0.05). Antibody against GST protein was used as a loading control.

Supplementary Figure 6. Flies producing Rab35DN have actin-related defects in

multiple tissues.

A, C, E, G, I, tissues from Rab35WT-expressing flies; B, D, F, H, J, tissues from

Rab35DN-expressing flies. Bristle kinks were seen in the head (arrows in B). Scale bar,

0.2 mm. Developing egg chambers had disorganized structures (D), fewer nurse cell actin

cables (F, shown by lower intensity actin staining), a thinner follicle cell layer (H), and fewer

actin cables in follicle cells (J, shown by lower intensity actin staining). Scale bars, 10 µm.

Note that in G, Rab35WT was localized near the plasma membrane and in intracellular

vesicles. In H, Rab35DN was less associated with the plasma membrane and more dispersed

in the cytosol.

Supplementary Figure 7. Fascin, but not Rab35, has actin-bundling activity in vitro.

GST-Rab35 and/or GST-fascin were incubated with non-muscle F-actin in conditions

previously found to stimulate actin bundling. After a low speed centrifugation to separate

bundled actin from filaments, aliquots of the supernatant (S) and pellet (P) were fractionated

on a gel.

A. Increased fascin concentration (0.1, 0.5, 1, 5, 10 µg) was correlated with increased

bundling activity (lanes 5&6, 7&8, 9&10, 11&12, 13&14). The actin filament bundling

protein α-actinin was used as a positive control (lanes 3 and 4). Providing additional

Rab35 (5 or 15µg) did not increase the bundling activity of fascin (lanes 15-18; compare

to lanes 9, 10).

B. More sensitive Immuno blotting of diluted samples (1:100) from A to show increased

actin bundling activity by fascin (1, 5, 10 µg) in lanes 3-8.

C. Efficient bundling is indicated by more actin (compare lanes 5, 6 to 1, 2), or more

actin plus α-actinin (compare lanes 5, 6 to 3, 4), in the pellet after centrifugation. Fascin has

bundling activity comparable to α-actinin (lanes 9, 10 compared to 5, 6). The addition of

Rab35 (either Rab35WT or Rab35DN) did not increase actin bundling (lanes 15, 16, 21, 22).

Rab35 did not co-centrifuge with actin (lanes 13, 14, 19, 20). The actin filament bundling

protein α-actinin was used as a positive control (lanes 5, 6).

Supplementary Figure 8. Rab35WT or DN localization in Drosophila S2 cells.

Rab35WT was localized in plasma membrane and intracellular structures. Rab35DN,

incontrast, was mostly located within cells away from the plasma membrane. Green,

Rab35; red, actin. Scale bar, 3 µm.

Supplementary Figure 9. Blocking fascin function reduces filopodia protrusions in cells.

A. Fascin shRNA produced in NIH3T3 cells for 3 days efficiently knocked down fascin

protein level (lane 3, compared to lanes 1 and 2). Tubulin level was shown as a loading

control.

B-C. NIH3T3 cells were transfected to produce Rab35 with (C) or without (B) fascin

shRNA. Dramatically reduced filopodia-like protrusions were observed in most cells that

produced fascin shRNA (C). Scale bars, 5 µm.

D-F. NIH3T3 cells were transfected to express Rab35 with fascinWT (D), fascinS39A

(E), and fascinS39D (F). In more than 50% of the cells, the activated form of fascin

(S39A) did not further increase the number of filopodia-like protrusions (E), but caused more

protein to accumulate at the tips of filopodia, compared to cells producing Rab35 and

fascinWT (D). The inactive fascin form (S39D) reduced the cell morphology changes caused

by Rab35 (F). Scale bars, 5 µm.

Supplementary Figure 10. FascinS39A and fascinS39D effects on Rab35-induced

filopodia protrusions in cells.

Here we provide additional examples of cells treated and stained as in Figure

S9D-F (A-C, D-F, G-I to S9D, 9E, 9F, respectively). Scale bars, 5 µm.

J. Quantification of filopodia numbers in fascinS39A and S39D-treated cells. FascinS39A in

combination with Rab35WT induced 83.6±18.29 filopodia compared to Rab35WT alone

(76.9±16.43, p>0.05). FascinS39D in combination with Rab35WT blocked

filopodia formation (11.0±2.98) compared to Rab35WT alone (p<0.01).

Supplementary Figure 11. Mitochondrial targeting of Rab35WT, but not Rab35DN,

triggers actin localization near mitochondria.

A-D. Additional examples of cells treated and stained as in Figure 4. A-B show cells where

Rab35WT was localized on mitochondria, causing actin (red) to accumulate near the

mitochondria (green). C-D show the control experiment with Rab35DN, where little to no

actin accumulated near mitochondria. Scale bars, 1 µm.

E. Model for Rab35 effects on actin bundling. 1. When the cell needs to extend

protrusions, Rab35 is activated by binding to GTP; the relevant GDP exchange factor

is not yet known. Activated Rab35 moves to the plasma membrane. 2. Active Rab35

binds and recruits its effector(s), one of them fascin, to the plasma membrane. Pre-

existing actin filaments are in the vicinity. 3. Rab35 in its active GTP-bound state

binds and concentrates fascin near the plasma membrane. The fascin induces actin

filament bundling by crosslinking filaments. In cultured fibroblasts, this leads to

filopodia formation. 4. When RNAi is used to reduce the amount of Rab35, little

activated Rab35 reaches the plasma membrane. In the absence of localized fascin and

other ABPs, actin filaments are not preferentially bundled near the plasma membrane.

Proper bundles do not assemble so in cultured cells no extensions are made, and in

flies the bristles are improperly formed. 5. The inadequate level of Rab35 caused by

RNAi treatment can be corrected by flooding the cells with fascin, restoring most

bristle morphology. Note the proteins are not represented to scale.

Supplementary Figure 12. Drosophila S2 cells treated with Rab35 dsRNA specifically

reduce Rab35 protein levels.

A. S2 cells producing GFP-Rab35, were treated with Rab35 dsRNA for 3-7 days. By the end

of five days, over-expressed and endogenous Rab35 proteins, which differ in size by virtue of

the GFP (appearing as two bands at 50 KDa and 22KDa), were not detectable by anti-Rab35

antibody applied to Immuno blots (lane 4 compared to the others). Tubulin level was shown

as a loading control.

B. Extracts of S2 cells producing GFP alone, GFP-Rab1, GFP-Rab11, GFP-mouse

Rab35 and GFP-fly Rab35 were loaded in lanes 1, 2, 3,4, 6. Drosophila Rab35 antibody

specifically detected endogenous Drosophila Rab35 and GFP-fly Rab35 (22KDa band and

50KDa band, lane 6), not GFP-mouse Rab35 (lane 4), GFP-Rab1 (lane 2), or GFPRab11

(lane 3). GFP signals were shown by blotting with an anti-GFP here. Tubulin level was

shown as a loading control.

A

1 2 3 4 5

Supplementary Figure 12

Rab35

Tubulin

GFP50KDa

27KDa

50KDa

22KDa

GFP GFP

-Rab

1G

FP-R

ab11

GFP

-mou

se R

ab35

GFP

-fly

Rab3

5

Rab3

5dsR

NA 5

d

GFP

-fly

Rab3

5

B

1 2 3 4 5 6

50KDa

22KDaRab35

Tubulin

GFP

GFP

-Rab

35G

FP-R

ab35

Rab3

5dsR

NA 3

dG

FP-R

ab35

Rab3

5dsR

NA 5

dG

FP-R

ab35

Rab3

5dsR

NA 7

d

Supplementary Table 1: Rab35 binding proteins indentified by Mass Spectrometry

Protein name

GI number Protein size

The mammalian gene collection (MGC) number

14-3-3theta 5803227 28KDa Rho GDI alpha 36037 30KDa 117248 Syntaxin binding protein 1 73760415 67KDa 10436 Dihydropyrimidinase related protein DRP-2 50811906 62KDa 64873 Fascin homolog 1, actin-bundling protein 49472815 55KDa 14372 RAP1, GTP-GDP dissociation stimulator 1 68226674 61KDa 118858 Calcineurin protein phosphatase 3 catalytic subunit, alpha isoform (PPP3CA)

142369518 59KDa

34395

Chaperonin containing TCP1, subunit 8 (theta) 48762931 58KDa 111375 Chaperonin containing TCP1, subunit 2 (beta) 5453603 58KDa 142076 Protein kinase C and casein kinase substrate 107KDa 34644 Actinin, alpha 4 isoform 2 12025678 100KDa 12692 Valosin containing protein 6005942 97KDa 148093 Rabaptin 31377798 100KDa 48839 Tumor rejection antigen (gp96) 4507676 92KDa 75130 Microtubule associated protein 2 119590863 202KDa 138549

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