Gene screen plus membranes

Protein and mRNA expression was determined by western and northern blotting, respectively (7 (link),9 (link),15 (link)). Endothelial cells were lysed in electrophoresis buffer (125mM Tris [pH 6.8], 12.5% glycerol, 2% SDS, and trace bromophenol blue) and proteins separated by SDS-polyacrylamide gel electrophoresis. Following transfer to nitrocellulose membranes, blots were blocked with phosphate buffered saline (PBS) and non-fat milk (5%) and then incubated with antibodies against cyclin D1 (1:500), cyclin E (1:500), cyclin A (1:500), phospho-Rb (1:100), p27(1:300), p21 (1:500), cyclin B1 (1:500), cdk1, phospho-cdk1 (1:100), or β-actin (1:200). Membranes were washed in PBS, incubated with horseradish peroxidase-conjugated secondary antibodies and developed with commercial chemoluminescence reagents (Amersham, Arlington Heights, IL). For p21 mRNA expression, total RNA was loaded onto 1.2% agarose gels and fractionated by electrophoresis. RNA was blot transferred to Gene Screen Plus membranes and then incubated overnight at 68°C in hybridization buffer (Amersham, Arlington Heights, IL) containing [³²P]DNA probes (1×10⁸ cpm) for p21 or GAPDH. After hybridization, membranes were washed, and exposed to X-ray film. Protein and mRNA expression was quantified by scanning densitometry and normalized with respect to β-actin or GAPDH, respectively.

Total RNA was isolated from endothelial cells with Trizol, loaded onto 1.2% agarose gels and fractionated by electrophoresis. RNA was blot transferred to Gene Screen Plus membranes and prehybridized at 68 °C for 4 h in rapid hybridization buffer (Amersham, Arlington Heights, IL). Membranes were then incubated overnight at 68 °C in hybridization buffer containing [³²P]DNA probes (1 × 10⁸ cpm) for HO-1 or 18S mRNA [31 (link),22 (link)]. DNA probes were generated by RT-PCR and labeled with α-[³²P]dCTP using a random primer kit (Amersham, Arlington Heights, IL) as previously described [31 (link),35 (link)]. Following hybridization, membranes were washed, exposed to X-ray film at −70 °C, and mRNA expression quantified by densitometry and normalized with respect to 18S rRNA.