Figure 3.

Conditioned medium from HEK293T cells expressing the ectodomain construct contains secreted TMEFF2 ectodomain and inhibits ERK phosphorylation. A: Exponentially growing HEK293T cells transfected with the TMEFF2-ectodomain construct or the empty vector (EV) as a control, were grown under serum starvation conditions for 24 hours. A sample of the conditioned medium was then collected, concentrated and subjected to immunoblotting with an anti-TMEFF2 antibody targeting the ectodomain region of the protein. The presence of TMEFF2 ectodomain in the conditioned medium is indicated by the arrow. B: RWPE-1 cells were transferred to basal KSF medium for 30 minutes before the medium was replaced with different amounts of ectodomain containing medium. Two different TMEFF2 antibodies were added to the conditioned medium to neutralize the TMEFF2 ectodomain. IgG was used as control. Whole cell lysates were prepared and subjected to immunoblotting with anti-p-ERK1/2 antibody. Representative examples of at least two independent experiments showing similar results are shown.