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Fig. 2. Confirmation and specificity of the PDZK1-Bcr interaction. GST-PDZ domain fusion proteins from PDZK1 (A) as well as EBP50 and GOPC (B) were bound to glutathione-Sepharose beads, incubated with Calu-3 cell lysate, and the bound products separated by SDS-PAGE followed by western blotting using the anti-Bcr C20 antibody. (C) Confirmation of expression of GST fusions with PDZ domains of EBP50 and GOPC. (D) Bcr was immunoprecipitated from lysates prepared from BHK-21 cells transiently expressing Bcr and FLAG-tagged PDZK1, separated by SDS-PAGE followed by western blotting. (E) FLAG-tagged PDZK1-M2 was transiently expressed in BHK-21 cells with or without Bcr fused to GST. Lysates prepared from these cells were incubated with glutathione-Sepharose beads and the material pulled down was separated by SDS-PAGE. PDZK1 was detected by western blotting with the mAb anti-FLAG M2. (F) Bcr, Bcr-V1271A and Bcr-delTEV were transiently expressed in BHK 21 cells and lysates prepared from these cells were incubated with PDZK1 PDZ 1-GST bound to glutathione-Sepharose beads. The bound material was separated by SDS-PAGE and Bcr was visualized by western blotting using the anti-Bcr N20 antibody.
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