Interaction of p65 with GMP. (A) Analysis of the p65–GMP complex formation by autoguanylation (i) SDS-10% PAGE and (ii) autoradiography. Lane M, molecular weight marker; lane 1, virion particle; lane 2, BSA and Lane 3, purified recombinant p65. Arrow indicates the position of the radiolabelled protein band (B) Effect of mutations on autoguanylation activity of p65. Lane 1, mutated (K21A) p65 protein; lane 2, wild type p65 protein; lane 3, mutated H93A protein; lane 4, virion particle of AmCPV; lane 5, mutated H105A protein. Arrow indicates the position of the radiolabelled protein band (C) Effect of pH on autoguanylation assay. (D) Effect of divalent ions in the formation of p65-GMP complex. (E) Analysis of the stability of p65-GMP complex by TLC: Lane 1, negative control; lane 2, NaOH treatment; lane 3, HCl treatment; lane 4, NH2OH treatment.