Anti cyclin dependent kinase 4

The cells were grown in complete medium on six-well plates to 70–80% confluence, after which they were exposed to normoxia (95% air and 5% CO₂) or hyperoxia (95% O₂ and 5% CO₂) for up to 48 h. In a separate set of experiments, the cells grown on six-well plates were treated with DMSO or 30 µM PD98059 for 24 h. Following these treatments, whole-cell protein extracts were obtained by using the radio immunoprecipitation assay lysis buffer system (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-24948) and subjected to western blotting with the following antibodies: anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-47778, dilution 1:1000), anti-cyclin A (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-751, dilution 1:1000), anti-cyclin D1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-8396, dilution 1:250), anti-cyclin-dependent kinase (Cdk) 4 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA; sc-23896, dilution 1:250), anti-p27 Kip 1 (Abcam, Cambridge, MA, USA ; ab32034, dilution 1:1000), anti-total ERK1/2 (Cell Signaling, Danvers, MA, USA; 4695, dilution 1:1000), anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 9106, dilution 1:1000). The immunoreactive bands were detected and quantified as described in the “in vivo experiments” section.