Fig. 2. RT-PCR showing the expression of NO_GCR subunit mRNA in rat cerebellar granule cells (A) and Western blots showing the presence of NO_GCR subunits in granule cell extracts (B). + and – indicate PCR reactions performed using equivalent amounts of RNA which had or had not (respectively) undergone previous reverse transcription. Western blotting shows the presence of NO_GCR ₁ and ß₁ subunits in granule cell extracts. 20 µg of soluble cellular extracts were electrophoresed and transferred to PVDF membranes. Blots were incubated with NO_GCR, polyclonal antiserum (1:1000) in blocking buffer overnight at 4°C with constant agitation (control), or in the presence of ₁ peptide (0.1 µg/ml), ß₁ peptide (0.2 µg/ml) or ₁ plus ß₁ peptide. Once washed (3x10 minutes), the blots were incubated with anti-rabbit-IgG:HRP (1:5000) for 1 hour at 37°C.