Figure 1.

Receptor-interacting protein 1 regulates p27^Kip1 levels. (A) Western blot shows that p27^Kip1 levels are increased in primary MEFs from RIP1^−/− mice compared with wild-type controls (RIP1^+/+ cells). Cells were cultured in DMEM with 10% FBS. (B) RIP1^+/+ and RIP1^−/− immortalized cells were serum starved for 72 h followed by western blot. (C) Introduction of RIP1 (using adenovirus) into RIP1^−/− MEFs prevents p27^Kip1 accumulation in response to serum starvation (48 h). In addition, RIP1 downregulates p27^Kip1 accumulated in response to serum starvation (96 h, RIP1 was added after 48 h of serum starvation). In RIP1 (−) lanes, tetracycline was added to prevent the expression of RIP1. (D) Overexpression of RIP1 in U87MG cells prevents the accumulation of p27^Kip1 in response to serum starvation for 48 h. RIP1 was introduced into cells at the onset of serum starvation. Densitometry values show relative signal intensity normalized for ERK2 (loading) signal. (E,F) Expression of RIP1 for 48 h does not affect (E) p21^Cip1 or (F) CDK4 levels in RIP1^−/− MEFs. (G) Expression of RIP1 in RIP1^−/− MEFs results in phosphorylation of Rb. (H) RIP1 influences p27^Kip1 at the mRNA level. Northern blot probed with a mouse probe for p27^Kip1 and also with a GAPDH probe to check loading. RIP1^−/− MEFs were infected with Ad-RIP1 overnight followed by RNA extraction and northern blot. Three independent experiments were performed and a representative experiment is shown. CDK4, cyclin-dependent kinase 4; ERK2, extracellular signal-regulated kinase 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MEF, mouse embryonic fibroblast; pRb, phosphorylated Rb; Rb, retinoblastoma protein; RIP1, receptor-interacting protein 1.