Figure 1

Manf localises intracellularly partially to ER and endosomal compartment. A-C - The confocal micrographs of 2nd instar larval garland cells stained for α-Manf (magenta) showing Manf expression around the nuclei (A) overlapping partially with ER-EYFP marker (green), DAPI (blue) was used to stain nuclei (A-C). D - Western blot analysis shows two fold increased amount of phosphorylated elF2α in Manf^mzΔ96 embryos in comparison to wild type w¹¹¹⁸ embryos. Decreasing amounts of samples were loaded to obtain the optimal result for quantification; the triangles represent the direction of decrease in loading. α-tubulin (α-tub) was used as a loading control. E-G - The confocal micrographs of Schneider-2 cells transfected with Manf cDNA construct and stained with Lysotracker (green) and α-DmManf show almost no colocalisation (less than 0.3%). H-M - The confocal micrographs of the wild type 3rd instar larval fat body expressing GFP-tagged UAS-constructs (green) driven by fat body specific ppl-GAL4 and stained for α-DmManf (magenta); nuclear stain DAPI (blue) was used. In H-J Manf localises close to clathrin coated vesicles marker GFP-clathrin light chain (Clc). In K-M Manf shows partial colocalisation with late endosomal compartment marker Rab7. N-S - In the salivary gland cells of 3rd instar larvae Manf (magenta) localises close to the basal cell borders and colocalises partially with early endosomal marker Rab5 (green) (N-P) and the recycling endosomal pathway marker Rab11 (green) (Q-S). Close arrows mark the cell borders and the open arrows mark the areas of colocalisation; all images consist of single laser confocal section. Scale bars: in A-C 2 μm, 4 μm in H-J, 5 μm in E-G and K-S.