Using the c-Jun (p-Ser73) assay kits, c-Jun phosphorylation at Ser73 is detected in anisomycin-treated NIH3T3 cells (+), compared with untreated cells (-), whereas no change in total c-JUN levels was observed.

Using the PhosphoTracer total-c-Jun assay, the expression of c-Jun was examined in several cell lines. Cells were seeded at 40K/well in 96-well microplates, and cultured overnight at 37°C in medium containing 10% FBS. The following day, the medium was removed from the wells, and the cells were lysed with 120 µL/well Lysis Mix, with shaking for 10 min. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and assayed for total c-Jun using the standard PhosphoTracer protocol. Signal in the wells was determined using a plate reader. c-Jun was readily detected in most cell lines, with the exception of MCF7 and A431 cells.

HeLa cells were seeded at 40K cells/well in medium containing 10% FBS in a 96 well microplate, and cultured overnight. The following day the culture medium was removed, and the cells were treated with various concentrations of anisomycin diluted in serum-free medium for 30 mins. The medium was removed from the wells, and the cells were lysed with 120 µL/well Lysis Mix, with shaking for 10 min. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and assayed for total c-Jun using the standard PhosphoTracer protocol. Signal in the wells was determined using a plate reader.