Fig. 2. Western blots confirm specificity of binding to zipcode and A2RE11. (A) 21-day-old rat brain proteins were used in pull-down experiments with magnetic particles bearing NS1, A2RE11 or 5' zipcode. The RNA-bound proteins were eluted, run on SDS/polyacrylamide gels and electroblotted. The blots were incubated with the indicated primary antibodies and protein bands detected with alkaline phosphatase-conjugated secondary antibody. The molecular masses in kDa are indicated on the left. (B) NS1-, AURE- and 5'-zipcode-binding proteins detected with anti-KSRP (C2742). (C) Western blots developed with chicken antibody to CRD-BP, which binds ZBP-1. On the left, proteins from embryonic (E15) and 21 day postnatal (P21) rat brains. No 68 kDa protein was observed in pull-down experiments with the A2RE or 5' zipcode and P21 chicken (centre) or P21 rat (right) brain protein. This blot was overdeveloped to show the weak band. (D) Western blot of rat brain proteins bound to NS1, A2RE11 and 5' zipcode detected with antibodies to conventional kinesin heavy chain. The positions of standard proteins are shown on the left, with molecular masses in kDa.