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Blood, Vol. 113, Issue 20, 4955-4962, May 14, 2009
CNTO 530 functions as a potent EPO mimetic via unique sustained effects on bone marrow proerythroblast pools
Blood Sathyanarayana et al.
113: 4955
Supplemental materials for: Sathyanarayana et al
Files in this Data Supplement:
- Figure S1. Differential activation of p70S6K and ERK pathways by CNTO 530 vs. EPO (JPG, 84.8 KB)
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(A) Primary bone marrow erythroid progenitor cells were expanded in SP34-EX, and KitposCD71highTer119neg pro-erythroblasts were isolated. Hematopoietic cytokines were withdrawn for 5.5 hours, and cells then were exposed to either epoetin-alfa (EPO) or CNTO 530 at doses / conditions in pilot experiments that similarly activated p70S6K (see below). At the indicated time points, lysates were prepared and analyzed for induced levels of phospho-T421/S424-p70S6K and phospho-T202/Y204-ERK1,2. (B) As illustrated by ratios of CNTO 530- vs. EPO- induced activation, each stimulated p70S6K phosphorylation at similar levels. By direct comparison, ERK activation by CNTO 530 was decreased approximately two-fold. In this experiment (exp. #1), integration of signals is for 15 and 45 minute time-points. (C) The above analyses were independently repeated, and outcomes were reproduced. In this experiment (exp. #2), integration is for a 20, 40, and 80 minute time-course. In each set of analyses, and for a given factor as induced by EPO or CNTO 530, a single common film was used for both phospho-STF antibodies within a single contiguous blot.
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