Figure 2

Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 (F, all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.