Supplemental figure legends
Figure S1. Specificity of phospho-S82 antibody is not affected by S79A
substitution or S79 phosphorylation.
The peptides shown in panel A were incubated with the pS82 antibody prior to Western
blotting of Flag-Cdc25A. Immunoblotting was performed with pS82 antibody preincubated with buffer (lanes 1, 2), A79-pS82 peptide (lanes 3 and 4), pS79-pS82 peptide
(lanes 5 and 6), S79-pS82 peptide (lanes 7, 8) or S79-S82 peptide (lanes 9, 10).
Figure S2. Specificity of phospho-S79 antibody is not affected by the S76A
substitution or S76 phosphorylation.
The peptides shown in panel A were incubated with the pS79 antibody prior to Western
blotting of Flag-Cdc25A. Immunoblotting was performed with pS79 antibody preincubated with buffer (lanes 1, 2), A76-pS79 peptide (lanes 3 and 4), pS76-pS79 peptide
(lanes 5 and 6), S76-pS79 peptide (lanes 7, 8) or S76-S79 peptide (lanes 9, 10).
Figure S3. Depletion of CK1 stabilizes Cdc25A.
HeLa cells were transfected with GL3 control siRNA (Ctrl) or siRNAs specific for CK1,
, , and 1. 48 h after transfection, cells were harvested and analyzed for the indicated
proteins by Western blotting. Anti-CK1 (C-19), anti-CK1(R-19), anti-CK1 (C-20) and
anti-CK1 (N-14) antibodies were obtained from Santa Cruz Biotechnology.
Figure S4. S82 phosphorylation is regulated by CK1.
(A) HeLa cells transfected for 20 h with plasmids encoding Flag-Cdc25A together with
plasmids encoding either CK1 or V5-LacZ were lysed and subjected to SDS-PAGE.
Proteins were analyzed by Western blotting for the indicated proteins. (B) HeLa cells
transfected with control siRNA (Ctrl) or siRNA targeting CK1 for 48 h were treated with
50 mM MG132 for 2 h and then harvested for Western blotting. (C) HeLa cells
expressing Flag-Cdc25A were cultured in the presence of DMSO (lane 1) or with
increasing concentrations of D4476 or IC261 for 1 h. Cell lysates were prepared and
analyzed by Western blotting. 25 M (lanes 2, 6), 50 M (lanes 3, 7), 75 M (lanes 4, 8),
100 M (lanes 5, 9). (D) HeLa cells transfected with plasmid encoding Flag-Cdc25A for
20 h were treated with DMSO, MG132 (50 M), D4476 (100 M), TBB (20 M) or KN-93
(10 M) for 60 min. Cells were harvested and analyzed by Western blotting for the
indicated proteins.
Figure S5. Conservation of human Cdc25A phosphodegron.
Human Cdc25A contains two motifs that regulate its interactions with -TrCP (pS79-T-DpS82-G and D215DGFVD220). These motifs are conserved in several higher eukaryotes
with the exception of Xenopus Cdc25A, which utilizes the DDGXD motif [PNAS
(2005) 102: 6279].