1. a 1 2 3 5 6 4 7 8 10 9 11 12
170 35 47 60 24 18 13 14 A B C D
Fraction number b
MCF7 lysate fraction A B C D A B C D
GST-p851
GST
Kinase assay
GST-p851
GST
Comassie blue staining
GST-p851
GST
Supplementary Figure S4. Glycerol gradient fractionation of MCF cell lysates to identify
the p85 serine 83 kinase.
a. MCF7 cell lysate was fractionated on a 3% to 15% glycerol gradient. After centrifugation,
14 fractions were collected and an aliquot of each fraction was separated by PAGE on
a 12% gel. Proteins were visualized by silver staining. Molecular weight markers are
indicated on the left. For n vitro kinase assays, fractions were combined as follows:
fractions 3 and 4 (A), fractions 5 and 6 (B), fractions 7 and 8 (C), and fraction 9 and 10 (D).
b. top: in vitro kinase assays using MCF7 cell lysate fractionated by a glycerol gradient
(A, B, C, and D) as the kinase source and GST fused p85 peptide spanning amino acids
(GST-p851) or GST only as substrates. Bottom: Comassie blue staining of
purified GST-p851 or GST showing equal protein amounts used in the kinase assay.