Overexpression of Dictyostelium GSK3

The GSK3 coding sequence was amplified from cDNA by RT-PCR using primers Pp-GSK3-S51 and Pp-GSK3-E31E (Additional file 1: Table 1) containing BglII and EcoRI sites, respectively. After cloning into pCR4-TOPO (Invitrogen) the PCR product was validated by sequencing, digested with BglII and EcoRI and cloned into BglII- and EcoRI-digested vector pDdNYFP [19 (link)], yielding vector pPp-A15GSK3-OE. To express GSK3 from its own promoter, the promoter region was amplified by PCR using primers Pp-GSK3-51 and Pp-GSK3-31 (Additional file 1: Table 1), cloned into pCR4-TOPO (Invitrogen) and sequenced. After digestion with SpeI and BglII, the 1.5-kb fragment, which contains the GSK3 promoter region, was cloned into NheI- and BglII-digested pPp-A15GSK3-OE. This yielded vector pPp-GSK3-OE, which was introduced into gsk3⁻ cells.