Figure 5.
mLin-7 binding did not influence delivery of receptor chimeras to the plasma membrane in a primary antibody capture experiment. MDCK cell lines expressing P75t-Let23WT (WT) or P75t-Let23MUT (MUT) were grown on Transwell filters, pulse-labeled with [35S]methionine for 1 h, and then chased for 3 h with complete growth medium containing antibody to P75 at either the apical (AP) or basolateral (BL) side to capture newly synthesized P75t-Let23 chimera. After washing to remove unbound antibody, the cells were lysed and the lysates subjected to precipitation with rabbit anti-mouse IgG and protein A-Sepharose. Precipitated proteins were separated by 10% SDS-PAGE and quantitated on a phosphorimager. Control precipitations were also performed to determine total labeled chimera protein. Delivery data is expressed as the fraction of captured P75t-Let23 divided by the total P75t-Let23. The experiment was performed in triplicate (n = 3) and the SDs are shown.