Figure 7

Expression of prostanoid receptors in HT-29 cells. A RT-PCR analysis of EP1-4 receptor expression in serum-fed HT-29 cells. See methods section and table 1 for details. Note that the primer pair employed in this experiment detects all known EP3 splice variants. B Serum-starvation induces EP3 mRNA levels. HT-29 cells were either kept in serum or serum was removed for the indicated number of days. EP3 expression was determined as in A. RT-PCR for GAPDH was run in parallel to control for equal loading. C Expression and regulation of EP3 splice variants. HT-29 cells were either kept in serum or deprived of serum for 24 h prior to performing RT-PCR on total RNA preparations. Primer pairs for individual EP3 splicing isoforms are listed in table 1. RT-PCR on total RNA from K562 cells was run as a control for EP3 isoform expression. As a further control HA-tagged EP3 subtype 3 was transfected into HT-29 cells prior to RT-PCR analysis. Note that EP3 subtypes 1–3 cannot be discriminated by use of different primer pairs but on the basis of the different size of the amplified fragments. A fragment amplified by the EP3 type 1/2/3 primer set of about 180 bp size that cannot be attributed to any known splice variant is marked by an asterisk. Control lane indicates an RT-PCR run using water as a template.