FIGURE 3:

Expression of GFP-CCTε as a probe for CCTε monomer function. (A) Construct design for expression of a GFP-CCTε fusion. (B) Lysate from BALB 3T3 cells expressing GFP-CCTε was fractionated on a 40–10% sucrose density gradient and then analyzed by SDS–PAGE and Western blotting to confirm that placement of the GFP tag renders the subunit monomeric. (C) GFP vector alone and GFP-CCTε localization in B16F1 cells 24 h posttransfection were compared. GFP-CCTε is found specifically to accumulate at the cell periphery and is quantified by counting the percentage of cells with peripheral staining from separate transfections (mean ± ­SEM, n = 3; *p < 0.05). (D) Staining of B16F1 cells with antibodies to tubulin and CCTε after stimulation with AlF₄ shows an accumulation of endogenous CCTε at the cell periphery, using the εAD1 monoclonal antibody. (E) Expression of GFP-CCTε but not GFP alone for 96 h results in increased cell spreading (***p < 0.001). Scale bars, 20 μm.