Supplemental Materials
Molecular Biology of the Cell
Jang et al.
Jang HI et al,
Supplemental Figures
Supplemental Figure 1. Mapping the interacting domains of VCP and Shoc2.
(A) Schematic representation of the Shoc2 truncated mutants used in the assays whose results are presented in panels B and C.
(B) 293FT cells were co-transfected with HA-VCP and the Shoc2-tRFP truncated mutants depicted in panel A. HA was immunoprecipitated and analyzed by immunoblotting using anti- HA and anti-tRFP antibodies.
(C) 293FT cells were co-transfected with VCP-GFP and the GST-Shoc2 truncated mutants depicted in panel A. GST was immunoprecipitated and analyzed by immunoblotting using anti- GST and anti-VCP antibodies.
(D) Co-immunoprecipitation of GST-tagged Shoc2 (WT and Δ12-14) and VCP-GFP. GFP and GST antibodies were used to detect VCP and Shoc2 in immunoprecipitates, and in total lysates of 293FT cells.
(E) Co-immunoprecipitation of GST-tagged Shoc2 (WT and Δ12-14) and HA-HECT. HA and GST antibodies were used to detect HECT domain of HUWE1 and Shoc2 in
immunoprecipitates, and in total lysates of 293FT cells.
WT- wild type, LRR- leucine rich repeat.
Supplemental Figure 2. VCP does not regulate the stability of RAF-1, Shoc2 or PSMC5.
(A) Cos1 cells were treated with different concentrations of CB-5083. At 4 hr after treatment, cells were harvested for immunoblotting. The expression of Shoc2, RAF-1, PSMC5 and GAPDH was analyzed.
(B) 293FT cells were transfected with HA-HUWE1 and GST-Shoc2 and subsequently treated with CB-5083. HA-HUWE1 was immunoprecipitated and analyzed by immunoblotting using anti-HUWE1 and anti-Shoc2 antibodies. Cell lysates were analyzed for HUWE1 and Shoc2.
Supplemental Figure 3. The ubiquitination of Shoc2 has no effect of Shoc2–PSMC5 complex localization on endosomes.
(A) Post-nuclear supernatants (PNS) from Cos1 cells stably depleted of Shoc2 and expressing WT Shoc2-tRFP or the 7KR mutant of Shoc2-tRFP were layered on 8-42% sucrose gradients
Jang HI et al,
and subjected to ultracentrifugation. Shoc2 was identified by immunoblotting (IB) using specific antibodies in PNS and crude endosome (CE) fractions.
(B) Cos1 cells stably expressing WT Shoc2-tRFP or the 7KR mutant of Shoc2-tRFP transfected with CFP-PSMC5 were followed by live-cell fluorescence microscopy. Scale bars: 10 μm.
Supplemental Figure 4. VCP controls levels of Shoc2 and RAF-1 ubiquitination
(A) Endogenous Shoc2 was immunoprecipitated from denatured cell lysates of Cos1 cells transfected with wild-type (WT) or catalytically inactive (QQ) mutant of VCP-GFP. Shoc2 ubiquitination was detected by immunoblotting using anti-ubiquitin (Ub) antibody.
(B) Endogenous RAF-1 was immunoprecipitated from denatured cell lysates of Cos1 cells transfected with wild-type (WT) or IBMPFD disease causative mutant (R155H). RAF-1 ubiquitination was analyzed by immunoblotting using anti-ubiquitin (Ub) antibody.
(C) Endogenous RAF-1 was immunoprecipitated from the denatured cell lysates of primary fibroblast using anti-RAF-1 antibodies. GM22757- normal control, GM23284- fibroblasts harboring VCP R155H mutation. RAF-1 ubiquitination was detected with anti-Ub antibody.
The results in each panel are representative of three independent experiments.
Supplemental Figure 1
IP:GST
IB:GFP
lysates
150 KD
IB:GST
IB:GFP
IB:GST VCP-GFP
GST-Shoc2
+ - - + +
100 KD
150 KD
100 KD
vector WT 12-14
WT 12-14
D.
HA-HECT GST-Shoc2
+
vector WT 12-14
IP:GST lysates
100 KD
50 KD
100 KD
50 KD
IB:HA IB:GST
IB:HA IB:GST
+ +
E.
A.
1 582
LRR
1 1 1
582 346
347
347 417 299
440
Shoc2-VCP binding
+ - - + + +
N-LRR9 N-LRR11 N-LRR15 LRR12-C LRR12-14
N C
1 582
LRR12-14
-
WT
B.
IP: HAlysates
IB:RFP
IB:HA
- + + + + + + HA-VCP
100 KD 75 KD
50 KD
100 KD
100 KD 75 KD
50 KD
100 KD
IB:RFP
IB:HA WT vectorWT N-LRR9
N-LRR11 N-LRR15
LRR12-C
C.
GST-Shoc2GST-Shoc2(LRR12-14) VCP-GFP GST
IB:VCP
IB:VCP IP: GSTlysates
+ + -
- - -
- - +
+ + +
+ +
- - -
-
- -
150 KD
150 KD
IB:GST 100 KD
75 KD 50 KD 37 KD
25 KD
Shoc2 LRR12-14 GST Shoc2
IB:GST 100 KD
75 KD 50 KD 37 KD
25 KD
GST Shoc2
Shoc2 LRR12-14
Supplemental Figure 2
IB:Shoc2 IB:HUWE1
IB:Shoc2 IB:HUWE1
IP : HAlysates
HA-HUWE1 GST-Shoc2
CB-5083
100 KD
250 KD
250 KD 100 KD
- + -
- + + + + + + - - IB: RAF1
IB: Shoc2
IB: GAPDH IB: PSMC5
50 KD 75 KD
50 KD
37 KD
DMSO 5 µM 10 µM
CB-5083
A. B.
Shoc2(WT)-G Shoc2(7KR)-G
B.
A.
IB:Shoc2
IB:EEA1 PNS CE
100 KD
WT 7KR WT 7KR
150 KD 250 KD
Supplemental Figure 3
G S T -P S M C 5
GST-PSMC5
Supplemental Figure 4
VCP-GFP vector WT R155H
IP:RAF-1
250 KD 150 KD 100 KD
75 KD
IB:Ub IB:RAF-1
IB:Ub
IB:RAF-1 IB:VCP
lysates
250 KD 150 KD 100 KD
75 KD 150 KD
B.
IB:Ub IB:RAF-1
IP:RAF-1lysates
250 KD 150 KD 100 KD
75 KD
GM23284 GM22757
IB:RAF-1
75 KD
IB:Ub
250 KD 150 KD 100 KD
C.
IP: Shoc2
250 KD 150 KD 100 KD
75 KD
IB:Ub IB:Shoc2
IB:Ub IB:VCP IB:Shoc2
lysates
250 KD 150 KD 100 KD
75 KD 150 KD
VCP-GFP vector WT QQ
