Supporting Information
Li et al. 10.1073/pnas.1017372108
A U2OS cell IP B HBL-100 cell IP
Input Ctrl anti-Siva1
Mr(K) Input Mr(K) Ctrl anti-Sta 18 Siva1 18 Stathmin Stathmin 18 18 Siva1 123 123
D pull-down
C MCF7 cell IP -Siva1
Input GST GST 18 His-Stathmin anti-Sta Mr(K) Input Ctrl 45 GST-Siva1 18 Stathmin Mr(K) 35 18 Siva1 GST 123 12 E G Siva1 Stathmin Stathmin Siva1 binding binding 148 114 145 175 16390 149 N DDHR RF ZF WT + NMCWT +
DDHR RF ZF N + MC -
N RF ZF DDHR + NC +
N DDHR C - NM +
RF ZF C + N +
H Flag-Siva1 + F GFP GFP-SIva1 GFP GFP-Stathmin
Mr(K) Mr(K) 45 45
35 35
IP: anti- 25 stathmin 18 Stathmin Flag-Siva1 IP: anti-Flag 45 45
35 35 Input Input 25 18 Stathmin Flag-Siva1 123456 123456
Fig. S1. Interaction of Siva1 and stathmin in vivo and in vitro. (A–C) Cell lysates from U2OS (A), HBL-100 (B), and MCF7 (C) were incubated with anti-Siva1 (A), anti-stathmin (Sta, B and C), or a control antibody. The input lysates and immunoprecipitated proteins were analyzed by Western blot. Molecular weight standards (Mr, in kilodaltons) are shown on the left. (D)His6-tagged stathmin was incubated separately with immobilized GST and GST-Siva1. Stathmin in the input and on beads (Upper) and GST protein on beads (Lower) were analyzed by Western blot and Coomassie blue staining, respectively. (E and G) Schematic representation of Siva1 (E), stathmin (G), and their deletion mutants. The amino acids at the domain junctions and the interactions with stathmin are indicated. Siva1 consists of N (N-terminal region), DDHR (death domain homology region), RF (B-box-like ring finger), and ZF (zinc finger-like domain). Stathmin consists of N (N-terminal), M (middle), and C (C-terminal) regions. (F and H) 293T cells were transfected with either GFP or the indicated GFP-Siva1 plasmid (F) and with GFP or the indicated GFP-stathmin fusion, plus Flag-Siva1 (H). Cell lysates were immunoprecipitaed with anti-stathmin (F)orFlag(H) antibody. Input and immunoprecipitated proteins were analyzed by Western blot.
Li et al. www.pnas.org/cgi/content/short/1017372108 1of2 A Siva1 CaMK II binding 148 114 145 175 N DDHR RF ZF WT +
DDHR RF ZF N -
N RF ZF DDHR +
N DDHR C +
N N +
B GFP GFP-SIva1
45 35 45 CaMK
IP: anti- CaMK 45 35 Input
45 CaMK 123 456
Fig. S2. Siva1 binds to CaMK II through its N terminus. (A) Summary of interactions of Siva1 and Siva1 deletion mutants with CaMKII. (B) 293T cells were individually transfected with empty vector, GFP-Siva1, or one of the Siva1 deletion mutants, and lysates were then immunoprecipitated with anti-CaMK II antibody, followed by Western blotting.
A D Siva1 siRNA -+ -+ Siva1 -WTC Stathmin siRNA -+-+
116 E-cadherin 116 E-cadherin 116 -Catenin 116 -Catenin Fibronectin 116 Fibronectin 116 Vimentin 45 Vimentin 45 Flag-Siva1 18 Siva1 18 Flag-Siva1- C 18 Stathmin GAPDH 35 GAPDH 35 12 3 123 4
B Siva 1 shRNA - + E-cadherin 116 C E-cadherin Vimentin Siva1 116 -Catenin Fibronectin
116 - Vimentin 45 18 Siva1
25 +
Flag-Siva1 Siva1 shRNA 35 GAPDH 12
Fig. S3. Siva1 inhibits EMT. (A and B) Expression of epithelial markers (E-cadherin and α-catenin) and mesenchymal markers (vimentin and fibronectin) in U2OS cells transiently transfected with control vector (−), Flag-Siva1, or Flag-Siva1 ΔC(A), or stably expressing a control shRNA (−) or Siva1 shRNA. (C) MCF-10A cells stably expressing the indicated shRNAs were analyzed for expression of E-cadherin and vimentin using immunofluorescence. (D) U2OS cells were transfected without or with Siva1 siRNA and/or stathmin siRNA. Protein expression was analyzed by Western blot.
Li et al. www.pnas.org/cgi/content/short/1017372108 2of2