He nucleus and in the cytoplasm surrounding the nucleus in HPAEC monolayer [4]. We investigated whether the RACK1-TIMAP complex formation has any effect on the subcellular localization of TIMAP. To modulate the interaction, HPAEC monolayers were subjected to agents affecting the phosphorylation level of TIMAP and the subcellular localization was detected by immunofluorescence studies of the monolayers or by Western blot of subcellular fractions (Figure 4A,B). Confocal images on Figure 4A show that the applied effectors did not change the cytoplasmic localization of RACK1 (Figure 4A b,e,h,k). On the other hand, upon forskolin treatment, the amount of nuclear TIMAP decreased parallel with its more pronounced appearance in the cell membrane (Figure 4A d) compared to the untreated sample (Figure 4A a). When cells were pretreated with a PKA inhibitor, H89, no translocation of TIMAP to the cell membrane was observed upon forskolin challenge, proving the involvement of PKA activity (Additional file 3: Figure S3). Since PKA phosphorylation of TIMAP on Ser337 primes its GSK3 phosphorylation on Ser333 [5,21], AR-A014418, a selective GSK-3 inhibitor [24], was employed alone or asBoratk?et al. Cell Communication and Signaling 2013, 11:2 http://www.biosignaling.com/content/11/1/Page 5 ofFigure 3 TIMAP-RACK1 interaction is attenuated by the cAMP/PKA pathway. (A) GST, full-length GST-TIMAP (upper part) or GST-RACK1 (lower part) were immobilized on glutathione-Sepharose and incubated with cell lysates of non treated (ctr), forskolin (50 M for 30 min) (FRSK) or PMA (1 M for 30 min) treated BPAEC. The eluted proteins were tested by Western blot using anti-RACK1 and anti-TIMAP antibodies. (B) Endogenous T3-Bromo-4-(propan-2-yloxy)aniline hydrochloride3-Bromo-4-(propan-2-yloxy)aniline hydrochloride title=View Abstract(s)">PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25386826 IP complexes were probed for TIMAP and RACK1. Shown are representative data of means ?SE from at least 3 independent experiments. Protein levels were quantified by densitometric analysis. Eluted proteins were normalized against total protein levels.pretreatment before addition of forskolin to prevent PKA primed phosphorylation of TIMAP by GSK-3. Without forskolin, no TIMAP was detected in the plasma membrane when GSK-3 was inhibited (Figure 4A g); also, the effect of forskolin was strongly attenuated in the (R)-1-(3-Chlorophenyl)ethan-1-ol presence of AR-A014418 (Figure 4A j). Merged images indicate colocalization of RACK1 and TIMAP in the region of cytoplasm that is rather close to the nucleus in control and GSK-3 inhibited cells cells (Figure 4A c,i,l), but colocalization was not detectable in the cells treated exclusively with forskolin (Figure 4A f). Membrane and nuclear fractions of HPAEC were isolated by cell fractionation as described in Materials and Methods and the amount of TIMAP in the fractions was detected by Western blot (Figure 4B). Parallel with the results of the immunofluorescent staining, the amount of TIMAP increased in the membrane fraction after forskolin, but it was significantly lowered in the presence of GSK-3 inhibitor compared to the control. Forskolin challenge in GSK-3 inhibited cells caused significant increase in the TIMAP level in the membrane fraction compared to the extremely faint PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 signal found in the same fraction of cells treated only with the kinase inhibitor. Furthermore, Western blot analysis of the nuclear fractions, as expected, demonstrated opposite trends for the changes. The least amount of TIMAP was detected in the nuclear fraction of the f.