(A) There were no substantial differences in the phosphorylation status of A-, B- and c-Raf in cyclin I–null (–/–) and WT (+/+) podocytes under nonstressed conditions using several phosphospecific antibodies. (B) Phosphorylation of MEK1/2 on residues Ser217/221 was substantially reduced in cyclin I–null podocytes (lane 2) compared with WT podocytes (lane 1) under physiological, nonstressed conditions. Restoring cyclin I levels in null cells by retroviral infection normalized MEK1/2 phosphorylation (lane 3) to levels comparable to those of WT podocytes. GFP transfection had no effect (lane 4). Total MEK served as a loading control. These results show that MEK1/2 Ser217/221 phosphorylation is cyclin I dependent. (C) Phosphorylation of ERK1/2 on residues Thr202/Tyr204 was reduced in 2 different clones of cyclin I–null podocytes (lanes 3, 4) compared with 2 different WT podocyte clones (lanes 1, 2). Restoring cyclin I levels in null cells by retroviral infection normalized ERK1/2 phosphorylation (lane 5) to levels comparable to those of WT podocytes; GFP infection had no effect. Total ERK1/2 served as loading control. These results show that the cyclin I–dependent activation of MEK1/2 was reflected by an increased phosphorylation of ERK1/2. (D) Reducing Cdk5 expression in cyclin I WT podocytes with siRNA decreased ERK1/2 phosphorylation (lane 2) compared with nontransfected cells. Scrambled siRNA had no effect on ERK1/2 phosphorylation.