CD98 Regulates Proliferation by Activating MAPKs Figure 5. CD98hc and Lat-1 increase cell proliferation. (A, B) Overexpressed Lat-1 interacts with CD98hc and CD98hc deletion mutants. (A) Western blot analysis of clones of IMCD cells transfected with Lat-1. Total Akt was used as a loading control. (B) Cells overexpressing CD98 or CD98 deletion mutants as well as Flag-tagged human Lat-1 were lysed and immunoprecipitated with a human specific CD98 antibody. Non-reduced (NR) and reduced (R) immunoprecipitates were separated by SDS-PAGE and immunoblotted with either a human specific CD98 antibody (upper panel) or Flag antibody (lower panel). (C, D) Co-expression of CD98hc and Lat-1 increases amino acid transport and cell proliferation. (C) 3H-Isoleucine uptake was performed as described in Experimental Procedures. IMCD/Lat-1cells are IMCD cells transfected with Lat-1 only and CD98/Lat-1 cells were the Lat-1 cells that were further transfected with CD98 and selected by flow cytometry. (*) denotes statistically significant differences (p,0.05) compared to IMCD cells whereas (**) indicates significant differences compared to CD98 cells. (D) Transfected cells were plated onto 96-well plates, serum deprived for 24 hours then treated for 16 hours with 3H-Thymidine in media containing 1% serum. 3H-Thymidine incorporation was determined as PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e40026