0 0.5 1 1.5
0 μM 0.1 μM 0.5 μM 2 μM MHCII-beta
alix
B
2006PS (μM) 0 0.1 0.5 2
C
MHCIIβ alix
UbP
A
100 kDa 80k Da
100 kDa 30kDa 28 kDa
2μM 2006PS (h) 0 1 6 24
MHCIIβ
UbP alix
MHCIIβ alix
UbP
**
*
0 0.5 1 1.5 2
0 h 1 h 6 h 24 h MHCII-beta
* alix
** *
*** ***
0 0.5 1 1.5
NS 2006PS R848 PolyI:C MHCII-beta
alix
*
*** ***
Relative band density (vs. 2006PS) Relative band density (vs. 2μM) Relative band density (vs. 24 h)
100 nm 49
6 kDa 98
28 SDS-PAGE silver staining 14
MHCIIβ flotillin-1
CD18
UbP
Fraction# 1 2 3 4 5 6 7 8 9 10 11
Density (g/ml) <1.03 1.03 1.05 1.07 1.09 1.12 1.16 1.20 1.26 >1.26 pellet
A B
400 μm
C
1.00E-20 1.00E-18 1.00E-16 1.00E-14 1.00E-12 1.00E-10 1.00E-08 1.00E-06 1.00E-04 1.00E-02 1.00E+00
0 5 10 15 20 25 30 35 40
45 Number of protiens
Fold enrichment P-value
C
B
NS 2006PSCD9 ++ ++
HSPA8 ++ ++
PDCD6IP - +
GAPDH - +
ANXA2 - ++
CD63 ++ +++
SCBP - +
ENO1 - +
EEF1A1 + ++
YWHAZ - +
ALDOA - ++
HSP90A
B1 ++ +++
ANXA5 + ++
CLTC ++ ++
CD81 - +
PPIA - ++
MSN + ++
PRDX1 - + RAP1B + ++
ITGB1 - + HSPA5 ++ ++
GNB2 - +
ATP1A1 ++ +++
FLOT1 ++ ++
CDC42 + ++
TUBA1B ++ +++
RAC1/2 ++ ++
34 132 223
NS 2006PS
A
Number of proteins/Fold enrichment P-value
0 200 400 600 800 1000
AB MV Exo
NS 2006PS
500g (cells)
1 500g (debris/AB)
10 000g (MV)
114 000g (Exo)
2006PS + - + - + - +
2000 1000 500 4000
200 28S 18S
A B
Protein (ng/μl)
* calnexin
actin UbP flotillin-1 MHC-Iiβ
LC3B-II alix
0 1000 2000 3000 4000
1 500g (AB) 10 000g (MV) 114 000g (Exo) NS
2006PS 500g
(cells)
1 500g (debris/AB)
10 000g (MV)
114 000g (Exo)
2006PS - + - + - + - +
RNA (ng/μl)
prohibitin-1 *
0 0.2 0.4 0.6 0.8 1 1.2
0 0.2 0.4 0.6 0.8 1
ASK CHSE-214 1.2
2006PS - + - + alix
flotillin-1 UbP
A
HKLs PBLs splenocytesB
2006PS - + - + - + alix
MHCIIβ
UbP MHCIIβ
Exo.
cells UbP
***
**** **
Relative band density (vs. HKL 2006PS) Relative band density (vs. 2006PS)
granulocytes
lymphoid cells
mononuclear phagocytes nd nd
alix alix
0 20 40 60 80 100 120 140
PBL HKL Fish 1 Fish 2
# adherent cells per viewfield
C D
Splenocytes PBLs HKLs ***
**
2006PS - + - + - + HKLs PBLs splen.
2006PS - + - + ASK CHSE
SSC
FSC
0 0.2 0.4 0.6 0.8 1 1.2
flotillin-1 UbP alix
Fraction# 1 2 3 4 5 6 7 8 9 10 11
Density (g/ml) 1.04 1.05 1.07 1.08 1.11 1.13 1.16 1.18 1.21 1.22 pellet alix
flotillin-1 UbP - 2006PS
Relative band density Relative band density
-
S. sal.
DNA
flotillin-1 UbP alix
flotillin-1 UbP alix
0 0.2 0.4 0.6 0.8 1 1.2
0 0.2 0.4 0.6 0.8 1
- 1.2
E. coli DNA
Relative band density
alix alix alix
** *** ***
A
B
S. sal. DNA - - + + DNase - + - + Ice + + - -
S. sal. DNA - + - +
Time (min) 5 min 45 min
S. sal. DNA - + - +
flotillin-1
UbP alix
adh.
post- adh.
S.sal. DNA Control
A C
flotillin-1 UbP alix
LC3B-II
D
B
flotillin-1
UbP alix
flotillin-1 UbP alix
LC3B-II
E
0 0.2 0.4 0.6 0.8 1 1.2
- SsDNA -
DNase
a a a b
Relative band density (vs. SsDNA)
alix
0 5 10 15
20 LC3B-II
alix
alix
LC3B-I LC3B-II LC3B-I LC3B-II long
exposure short exposure
a a a b
a b
a a
LC3B-Alexa555 (blue)
Alix-Alexa488 (yellow)
Orthogonal view
Orthogonal view
100 nm 100 nm
100 nm 100 nm
*
*
*
A
Relative band density (vs. 2137PS exosomes)
B C
2137PS - + - + 10 000 g 114 000 g WCL
10 000g (MV)
114 000g (exosomes) - 2137PS - 2137PS
A
flotillin-1
UbP alix
- 2395PS S. sal.
DNA
flotillin-1
UbP alix
B
S.sal. DNA - + +
DNase - - +
Relative band density (vs. heparin) alix flotillin-1
0 0.2 0.4 0.6 0.8 1 1.2
- heparin -
PD184352
0 0.2 0.4 0.6 0.8 1 1.2
- heparin a a
b b
a b c
d - - S. sal. DNA Heparin
PD184352 - + - + - +
Phospho-ERK flotillin-1
UbP exosomescell lysates
alix
Ponceau staining
