Fig 4.

(A) RNase A protection assay (RPA) of rat MC4R in penile tissues. Poly(A)⁺ RNA (6 μg) was hybridized with a rat MC4R cDNA probe (second intracellular loop to mid transmembrane-6) that, upon RNase digestion, would result in a radiolabeled fragment of 313 nucleotides, revealed by denaturing PAGE and autoradiography (as detailed in Materials and Methods). An internal standard rat MC4R fragment of 240 nucleotides is shown. (B) Binding of [¹²⁵I]MTII to rat penile membranes. Inhibition of [¹²⁵I]MTII binding by MTII and the THIQ MC4R agonist was performed by using 70 pM radiolabeled MTII for 70 min at 20°C; the results are expressed as percent of [¹²⁵I]MTII specifically bound. IC₅₀ values for MTII and MC4R agonist were 1.5 and 11 nM, respectively (n = 3 experiments).