Fig. 2. Immunoprecipitation of GFP fusion protein complexes. Anti-GFP immunoprecipitation was performed in lysates of stably transfected U1242MG cells expressing EGFP-p11, EGFP-AnxII or EGFP. (Top-left) Western blot for p11; 10% gel transferred to nitrocellulose. The p11 mAb recognized both endogenous p11 and EGFP-p11 in the lysates. Co-immunoprecipitation of p11 was seen only with GFP-p11. (Bottom-left) The same experiment as above; 15% gel transferred to PVDF in buffer containing 30% methanol. This procedure allows separation of p11 from the dye front and maximizes transfer of p11 to the membrane. (Top-right) Western blot for AnxII; 10% gel transferred to nitrocellulose. The AnxII mAb detects EGFP-AnxII only in the immunoprecipitates, since its apparent level in the lysates is much lower than endogenous AnxII. Co-immunoprecipitation of AnxII was seen only with EGFP-p11. (Bottom-right) Coomassie Blue stained gel shows distinct bands corresponding to EGFP and EGFP-AnxII in the immunprecipitates, whereas the much fainter EGFP-p11 reproduced poorly.