FIGURE 4.

Discovering and validating PTB binding specificity. (A, lanes 1–5) A Western blot of PTB immunoprecipitated from HeLa nuclear extract (N.E.) with (lanes 4,5) or without (lanes 2,3) mab BB7. (sup) Lanes containing IP or mock IP supernatant. (Lanes 6–8) Semi-quantitative PCR of oligonucleotides from the pool that co-immunoprecipitated with PTB. (Lanes 7,8) Oligonucleotides co-IP'ed in the absence of mab BB7 antibody or HeLa nuclear extract, respectively. (B) Motif finding on the top 1% of enriched oligonucleotides, using Gibbs sampling (Materials and Methods), resulted in a PTB consensus motif. (C, lane 1) UV-cross-linking to a high affinity ligand isolated from 11 rounds of SELEX (S¹¹) was performed on HeLa extract (Perez et al. 1997b). (Lane 2) The cross-linked extract was then immunoprecipitated using mab BB7 αPTB antibody. Samples were separated by PAGE. (Lanes 3–18) UV-crosslinking was performed using radiolabeled S¹¹ as a probe in the presence of increasing amounts (0-, 5-, or 50-fold molar excess) of unlabeled competitors as indicated. S⁷ is a PTB ligand isolated from seven rounds of SELEX (Singh et al. 1995). (E and E_Mot) Oligonucleotides associated with high enrichment scores; (E_Mot) also enriched for the motif described above; (Pool) the unenriched oligonucleotide pool.