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Blood, Vol. 107, Issue 3, 1133-1140, February 1, 2006
Tyrosine phosphorylation modulates binding preference to cyclin-dependent kinases and subcellular localization of p27Kip1 in the acute promyelocytic leukemia cell line NB4
Blood Kardinal et al.
107: 1133
Supplemental materials for: Kardinal et al, Vol 107, No 3, 1133-1140
Specificity of antibodies to p27Kip1
In our experiments, we used anti-p27Kip1 antibodies from 2 commercial sources (polyclonal: sc-528, from Santa Cruz, Heidelberg, Germany; and monoclonal: 610242, from BD Biosciences, Heidelberg, Germany). Their quality has been confirmed by Western blotting using total lysates of p27Kip1-null mouse embryonic fibroblasts (MEFs p27—/—), wild-type fibroblasts (MEFs wt; both types of fibroblasts were gifts from R. Medema, Amsterdam, The Netherlands), and NB4. Detection of the Western blot with both antibodies revealed strong reactivity in the p27Kip1 molecular weight range in MEFs wt and NB4 cells. As expected, no signal was detected in MEF p27—/— cells. As a p27Kip1-independent control, the Western blot was detected with an antibody to p21Cip1, which, like p27Kip1, belongs to the family of cyclin-dependent kinase inhibitors (Figure S1A).
Files in this Data Supplement:
- Figure S1. Specificity of antibodies to p27Kip1, evaluation of interaction of GST-p27Kip1 with cyclins, and phosphoamino acid analysis of 32P-labeled p27Kip1 (PDF, 1,776 KB) -
(A) A total of 100 µg MEF p27—/—, MEF wt, and NB4 lysate was detected by Western blotting with antibodies to p27Kip1 and p21Cip1. (B) GST-p27Kip1 wild-type (wt) fusion protein was expressed in Escherichia coli strains DH5
 (pTyr—: not tyrosine phosphorylated) and TKX1 (pTyr+: tyrosine phosphorylated). GST pull-downs from NB4 lysates were subjected to Western blotting and were analyzed for cyclin E and cyclin D. A total of 50 µg NB4 lysate was loaded as a control. (C) Immunoprecipitated 32P-labeled p27Kip1 polypeptide bands were cut out of the SDS-PAGE gel and subjected to phosphoamino acid analysis, using acidic hydrolysis followed by 2-dimensional chromatography as described.28 The phosphoamino acids are phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr).
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