CD98hc and Lat-1 increase cell proliferation.

<p>(A, B) Overexpressed Lat-1 interacts with CD98hc and CD98hc deletion mutants. <b>(A)</b> 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 described in Experimental Procedures. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p<0.05) compared to IMCD cells. (**) denotes a statistically significant difference between CD98 and CD98/Lat-1. (E) Cells expressing CD98 deletion mutants and Lat-1 proliferated less than CD98/Lat-1 cells. IMCD cells overexpressing Lat-1 were transfected with either CD98 or CD98 deletion mutant constructs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040026#pone-0040026-g001" target="_blank">Fig.1</a>. Thymidine incorporation was determined as described above. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (*)indicates statistically significant differences (p<0.05) between double transfectants and cells transfected with CD98 or deletion mutants only.