Fig. 6. Wnt proteins form homodimers. (A) Western blot analysis of gastrulae injected at the two-cell stage with 0.5 ng or 1 ng of Wnt11-HA mRNA. Lanes 1 and 2, no reducing agent; bands of Wnt11-HA are present at 50 kDa (monomer) and 100 kDa (dimer). Lanes 3-5, β-ME added (1, 5, 10%); only the 50 kDa band is visible. (B) Western blot of 125, 250 or 500 pg Wnt11-HA mRNA injected embryos+iodoacetamide (IAA, 10 mM). There is a 100 kDa band (^*) in the 250 and 500 pg lanes both in the absence (lanes 2, 3) and presence (lanes 5, 6) of IAA. β-ME converts to monomers (lanes 7, 8). (C) Western blot of 0.25, 0.5, 1 ng Wnt11-HA, Wnt8-HA or Wnt5a-Myc mRNA-injected embryos with IAA-containing buffer and non-reducing electrophoresis. Wnt11 (lanes 1-3), Wnt8 (lanes 4-6) and Wnt5a (lanes 7-9) form dimers. (D) Anti-GFP western blot analysis of embryos injected with 100 pg of Wnt11-GFP mRNA alone or +50, 100, 200, 500 pg of Wnt5a-Myc mRNA; non-reducing conditions. Wnt11-GFP alone (lane 1) produces a 75 kDa band (monomer) and a 150 kDa band (dimer). Co-injection with increasing doses of Wnt5a mRNA (lanes 2-5) showed no band of the expected 125 kDa (^*) of Wnt5a-Myc/Wnt11-GFP heterodimer. Lanes 6 and 7 show that the signal in lanes 1-5 is specific to Wnt11-GFP. Arrowhead indicates a non-specific band. (E) Anti-HA WB in non-reducing conditions where 0.5 ng of Wnt11-HA mRNA alone (lane 1) produced 50 and 100 kDa bands. Co-injection with 0.5 ng of Wnt11C-Flag mRNA (30 kDa) (lane 2) did not produce the 80 kDa band (^*) expected for Wnt11C-Flag/Wnt11-HA heterodimer. Similarly, the pattern of Wnt5a-Myc bands (lane 6) was not changed by the co-injection of Wnt11C-Flag (lane 4); ^*expected positions for a Wnt5a-Myc/Wnt11C-Flag heterodimer (80 kDa). ^**Wnt11C-Flag homodimer. No heterodimer bands were detected in co-injection samples (lanes 7, 12, asterisks) compared with Wnt11C-Flag alone (lane 8). Arrowheads indicate non-specific bands. (F) Analysis of Wnt11-HA and Wnt5a-Myc Co-IP under non-reducing conditions (NR) shows that only dimers (100 kDa) and oligomers (>100 kDa) of Wnt11-HA were precipitated by Wnt5a-Myc (lane 3); under reducing conditions (R), the IP product was seen a single monomer band (lane 4). (G) Western blot under non-reducing conditions of control and Dkk1-depleted oocytes injected with 1 ng Wnt11-HA or Wnt5a-Myc mRNAs. Dimer forms of Wnt proteins are marked with asterisks. Repeated experiments showed similar results.