Fig. 6
WNT5A is present in lipoprotein complexes. a CM from HbChP primary culture was subjected to KBr gradient-based ultracentrifugation and different isolated fractions (VLDL, LDL, and HDL) were analysed for the presence of WNT5A, APOE and APOJ by western blot, n = 3. (b–e) WNT5A-V5 and HA-tagged APOJ (b, d) or APOE (c, e) were overexpressed in HEK293T cells. WNT5A-V5 (b, d) and APOJ/E (c, e) were immunoprecipitated using anti-V5, anti-HA antibody and control IgG, respectively, and detected by western blotting, n = 3. Input is the loading control. Asterisk indicates non-specific immunoglobulin light chain. f Recombinant WNT5A (rcWNT5A) interacts with isolated APOJ protein. WNT5A and APOJ co-immunoprecipitated together. IgG served as a control, n = 3. Input represents the initial mixture. g Schematic depiction of the experimental design for delipidation and rescue experiments. h Delipidated FBS (Delipid) prevents production of WNT5A that is restored by relipidation (Relipid). Presence of WNT5A in cell lysates and CM from primary HbChP cultures has been analyzed using western blot, n = 3. Loading control: β-actin. i WNT5A secretion was restored after the addition of different mouse lipoprotein fractions to primary HbChP cultures cultivated in presence of delipidated conditions. j Recombinant rcWNT5A was incubated with either 0.6% CHAPS or human LDL (hLDL)- lipoprotein fraction for 4 h at 37 °C and filtered through 150 kDa cut-off protein concentrator. Western blot confirmed presence of WNT5A only in >150 kDa fraction but not in the <150 kDa fraction, n = 3. k Separation of >150 kDa fraction into VLDL, LDL, and HDL fractions using KBr gradient confirmed presence of rcWNT5A in LDL and its co-fractionation with APOB and APOE specific for LDL fraction, n = 3. l Only >150 kDa fraction of LDL/rcWNT5A mixture can trigger DVL3 phosphorylation (indicated by arrowhead) in MEF cells, n = 3. Negative control: 10% FBS DMEM; positive control: rcWNT5A; loading control: β-actin. m Statistical analysis of DVL3 shift assay. Graph shows n = 3 biologically independent samples; error bars represent mean ± s.d.; P-values (two-tailed Student’s t-test with unequal variance): * P < 0.05. Control CM vs >150 kDa hLDL + rcWNT5A: P = 0.0231. Biological replicates are indicated in the graph. Source data are provided as a Source Data
