Figure 2
LXN is cytosolic and also secreted. (A) Western blot analysis from subcellular fractionation studies in three prostate epithelial cell lines to determine LXN subcellular localisation. Representative blot from four independent experiments/cell line. α-tubulin and TBP indicate the purity of the cytosolic and nuclear fractions respectively. W.C (whole cell lysate), C (cytosolic), N (nuclear). See Supplementary Fig. S7 for uncropped original blots at 3 different exposures. (B) Confocal microscopy depicting Immunocytochemistry of PNT1A cells following transient overexpression of LXN-HA (upper panel). α-tubulin (middle panel) and TBP (lower panel) demonstrate the unbiased accessibility of both the nuclear and cytosolic compartments using this approach (Scale bar = 20 µM). (C,D) in silico analysis using SignalP 4.0 software to predict the occurrence of signal peptides in protein amino acid sequences reveals that LXN does not contain a predicted Signal peptide, whereas CPA4 does. (E) Nanoparticle tracking analysis (NanoSight NS300, Malvern) of positive control beads consistent with the size of exosomal particles (110 nM diameter) to determine that EVs purified were consistent with the size of exosomes in (F,G,H and I) which represent the purified EV fractions from PC3 and PNT1A cells respectively. (J) Enrichment of LXN from cell culture conditioned media following immunoprecipitiation using a LXN specific antibody compared to an isotype control and conditioned media control (input). This gel has been cropped to bring different parts of the same gel together for concisness and clarity. See Supplementary Fig. S2 for the full blot with all additonal controls. (K) Western blots demonstrating the relative abundance of LXN within purified EVs versus soluble secreted fractions (EV depleted media). TSG101 serves as a marker for successful isolation of endo/lysosomal material from the extracellular space. (L) Western blot of EV depleted media at two separate time points in PNT1A or PC3 cell lines. Tubulin was used to demonstrate the absence of any contaminating cellular material. Full length uncropped blots for (K,L) can be viewed in Supplementary Fig. S8A,B.
