14-3-3 

1. P-Akt 14-3-3 Control siRNA 14-3-3 siRNA Neal et al.Supplementary Figure S1 a b Control siRNA 14-3-3ζ siRNA C1 C2 si1 si2 si3 P-Akt Akt 14-3-3 c d Control siRNA 14-3-3 siRNA Control shRNA 14-3-3 shRNA1 14-3-3 shRNA2 0 30 45 0 30 45 0 30 45 0 20 30 0 20 30 minutes minutes P-Akt P-Akt Akt Akt 14-3-3 14-3-3 MDA-MB-435 MDA-MB-231 Supplementary Figure S1. Knockdown of 14-3-3ζ reduces Akt phosphorylation. a. MDA-MB-435 cells were transiently transfected with control or 14-3-3 siRNA. Forty-eight hours post-transfection 5 x 106 cells were injected into the mammary fat pad of SCID mice. Tumors were harvested 6 days post injection, fixed in formalin and embedded into paraffin. Tissue sections were stained for 14-3-3 and phospho-Akt-S473 (P-Akt). Nuclei were counter-stained blue with hematoxylin. b. Control siRNA (C#) and 14-3-3ζ siRNA (si#) tumor xenografts treated as in (A) were collected for western blot analysis. 14-3-3 and phospho-Akt -Ser473 (P-Akt) specific antibodies were used for detection. Dashed line indicates samples were on same gel but not consecutive lanes. c. MDA-MB-435 cells stably transfected with control or 14-3-3 shRNA were starved for 18 hours (0) and re-stimulated with 8% FBS for the indicated times (minutes). Akt activation was assessed by immunoblot for phospho-Akt -Ser473 (P-Akt). d. MDA-MB-231 cells transiently transfected with control or 14-3-3 pool siRNA were starved for 18 hours (0) and re-stimulated with 8% FBS for the indicated times (minutes). Akt activation was assessed by immunoblot for phospho-Akt -Ser473 (P-Akt).