Skip to main content
. 1998 Aug;18(8):4526–4536. doi: 10.1128/mcb.18.8.4526

FIG. 3.

FIG. 3

Specific association of cyclin E-cdk2 with SAP 155. Immunoprecipitation and Western analysis of SAP 155 association with cyclin E-cdk2 in ML-1 lysates. (a) In the left panel, an immunoprecipitation with the cyclin E-specific monoclonal antibodies HE67, 111, 135, 170, and 172 (lanes 2 through 6) is shown. Control immunoprecipitations were performed with an equivalent amount of coupled monoclonal antibody Pab419 (lane 1). All lysates were from ML-1 cells. Lysate control lanes contained 100 μg of cellular protein (lane 7). IP, immunoprecipitation. In the right panel is shown an epitope mapping of cyclin E-specific monoclonal antibodies. Peptides of the indicated overlapping sequences were synthesized onto paper (Research Genetics) and probed with the respective antibodies listed on the righthand side. (b) Detection of SAP 155 in HE172 immune complexes from ML-1 lysates depends on the presence of cyclin E protein in lysates. Cell lysates were precleared with the indicated antibodies three times. SAP 155 (upper panel) and cyclin E (lower panel) were detected in precleared lysates (lanes 3 and 4) or in cyclin E, and control antibody immune complexes were prepared from precleared lysates (lanes 2 and 1, respectively). (c) SAP 155 complexes were precipitated from lysates by using rabbit polyclonal SAP 155-specific antibody (lane 2). Coprecipitating cyclin E was detected with the cyclin E-specific monoclonal antibody HE12. The arrow indicates the top band of cyclin E detected in SAP 155 immune complexes. NRb, normal rabbit serum (lane 1). (d) Detection of SAP 155 associated with cdk2 (lane 2). NRb, normal rabbit serum (lane 1). (e) cdk2 associated with SAP 155 (lane 2) was detected by using antibody Sc-163 (Santa Cruz Biotechnology). NRb, normal rabbit serum (lane 1). (f) Absence of SAP 155 from cyclin A-specific immune complexes. Cyclin E (lane 3), cyclin A (lane 2), and control antibody immune complexes were tested for the presence of SAP 155 (top panel) and cdk2 (lower panel) by Western analysis. (g) SAP 155 associated with cyclin E complexes in different cell lines. Negative control, Pab419 (lanes 1, 2, 5, and 7); cyclin E-specific antibody, HE172 (lanes 2, 4, 6, and 8). SAP 155 detection was assessed by Western analysis by using polyclonal rabbit SAP 155 serum.