Figure 2

Cα2 associate with both RIα and RIIα in a cAMP-dependent manner in sperm cells. Sperm cell extracts were immunoprecipitated (IPed) with anti-RIα (1:100) and anti-RIIα (1:100). IPed proteins were separated by SDS-PAGE in 12. 5% gels, transferred to PVDF filters which were incubated with anti-Cα2 (1:20, upper panels) and a pan C antiserum (anti-Cγ (scbt) lower panels). Immunoreactive proteins which had been IPed (lanes 3-6 and lanes 9-12) were compared to immunoreactive proteins in the supernatant after the IP (lanes 2 and 8, S_IP) and to total input (lanes 1 and 7, Lysates). Cell extracts IPed with anti-RIα and anti-RIIα and sepharose beads coated with anti-IgG, were centrifuged and the pellets washed with a buffer with (+cAMP) or without (-cAMP) cAMP. Washed pellets (P) and the respective supernatants (S) were analyzed by SDS-PAGE and immunoblotting. Lanes 2 and 8 show anti-Cα2 immunoreactive proteins left in the supernatants after anti-RIα and RIIα IP (S_IP). Lanes 3 and 9 show anti-Cα2 immunoreactive proteins in supernatant of cAMP treated IP pellets. Lanes 4 and 10 show anti-Cα2 immunoreactive proteins in IP pellet after treatment with cAMP. Lanes 5 and 11 show anti-Cα2 immunoreactive proteins in supernatants of non-treated IP pellets. Lanes 6 and 12 show anti-Cα2 immunoreactive proteins in untreated IP pellet. Arrows on the left indicates migration of the molecular weight markers. Arrows on the right indicate the identity of the C subunit.