Fig 2: N-terminally tagged Wnt3a secretion from HEK cells.(A) Indicated amounts of CM from the confluent L cells stably expressing untagged Wnt3a (L-3a) or HEK293S GnT1- cells stably expressing TARGET-tagged Wnt3a (Tg-Wnt3a/HEK) were subjected to a Western blotting using anti-mouse Wnt3a antibody. Note that Tg-Wnt3a migrate slower than the untagged Wnt3a due to the presence of extra 35-residue (~4 kDa) tag sequence. (B) The stable TCF reporter cells were incubated with the indicated concentration of CM for 6 hr. Luciferase activities in the cell lysates were determined and expressed as the relative increase from the control value obtained in the mock-treated cells. Data are mean ± SD of three independent experiments, in which quadruplicate determinations were made. See also Figure 1—source data 1.DOI: http://dx.doi.org/10.7554/eLife.11621.00310.7554/eLife.11621.004Figure 1—source data 1.The Excel spreadsheet source file for Figure 1B.DOI: http://dx.doi.org/10.7554/eLife.11621.004

Fig 3: Affinity purification of tagged Wnt3a from the culture supernatants.A small column of Sepharose coupled with anti-TARGET tag antibody P20.1 (~3 ml) that had been incubated with ~220 ml of CM from the confluent culture was washed with TBS, followed by elution with TBS containing 0.2 mg/ml competing C8 peptide. Ten µl sample from each fraction (fraction size = 3 ml) was subjected to 5–20% SDS-PAGE under nonreducing condition and stained with Coomassie Blue. A portion of the same gel (fractions 7–11) was stained with Oriole fluorescent stain to visualize minor contaminating bands.DOI: http://dx.doi.org/10.7554/eLife.11621.005

Fig 4: Serum afamin binds to Wnt3a secreted from cells.(A) Serum-containing CM from the mock, L-3a, or Tg-Wnt3a/HEK cells were incubated with Sepharose beads immobilized with an anti-bovine AFM monoclonal antibody (clone B91) and the immunoprecipitated materials were separated by SDS-PAGE under reducing condition, followed by Oriole Fluorescent protein stain (left) and anti-Wnt3a immunoblotting (right). Positions for bovine AFM and tagged/untagged Wnt3a are shown in the right. Asterisks denote nonspecific serum-derived proteins. (B) L-3a or Tg-Wnt3a/HEK cells were cultured in DMEM containing various concentrations of serum or purified recombinant human AFM for 5 days, and the resultant CMs (3 µl) were subjected to the anti-Wnt3a immunoblotting. (C) Effect of various albumin family proteins to support Wnt3a secretion from L-3a cells is evaluated as in (B). HSA, human serum albumin; AFM, human afamin; AFP, human α-fetoprotein; VDBP, mouse vitamin D binding protein.DOI: http://dx.doi.org/10.7554/eLife.11621.006

Fig 5: Large scale production of Wnt3a and Wnt3 by using AFM co-expression method.(A) A 300 ml culture of Expi293F cells were used to co-express Tg-tagged human AFM and PA-tagged mouse Wnt3a or human Wnt3. The AFM-Wnt complexes were purified from the co-transfected culture media using NZ-1-immunoaffinity resin and checked for its purity on a nonreducing 5–20% SDS-PAGE gel followed by Coomassie Blue staining. (B,C) SEC profile of the NZ-1-purified AFM-Wnt3a (B) or AFM-Wnt3 (C) complex. The eluted material from the NZ-1 immunoaffinity chromatography (input, same as the sample shown in (A)) was subjected to the SEC on Superdex 200, showing two peaks. The void peak (fraction I) comprised only of HMW aggregates that barely enter the top of separation gel, while the ~140-kDa peak (fraction II) contained AFM and Wnt3a/Wnt3 at similar amounts. (D,E) Signaling activity of AFM-Wnt complex prepared by AFM co-expression method. The NZ-1-purified AFM-Wnt3a (D) or AFM-Wnt3 (E) complex was subjected to the TCF reporter assay as in Figure 5A. The data are mean ± SD of three independent experiments, in which quadruplicate determinations were made. See Figure 8—source data 1.DOI: http://dx.doi.org/10.7554/eLife.11621.02210.7554/eLife.11621.023Figure 8—source data 1.The Excel spreadsheet source file for Figure 8D and E.DOI: http://dx.doi.org/10.7554/eLife.11621.023