Probing LRP6 Interactions via Coimmunoprecipitation

(i) Membrane-anchored LRP6. P114-L2/L-HA cells were seeded in 15 cm tissue culture dishes. The next day, cells were washed with cold PBS and lysed in RIPA buffer containing protease inhibitor (cOmplete mix, Roche). The cell lysates were transferred into tubes, passed through a 26 G size needle (0.45 × 25 mm BL/LB, B Braun), and centrifuged at 4°C at 3,200 × g for 20 min. The supernatants were transferred into new tubes. In separate tubes, anti-TST Ab (THE NWSHPQFEK-tagged mouse MAb, A01732, GenScript, 1:150) was mixed with 20 μL of magnetic beads (Dynabeads Protein G, Invitrogen) in 300 μL of PBS containing 0.05% Tween 40 (PBS-T, Merck) and was incubated at 4°C for 10 min. Next, 15 μg of soluble HOP-TST, Hwt, HOP^A393T-TST, and Hwt^R529A-TST proteins were added to the Dynabeads/Ab mixture and incubated for 30 min on a rotating wheel at 4°C. The H protein/Ab/Dynabeads complexes were then mixed with the harvested supernatants and incubated on a rotating wheel overnight at 4°C. After five washes with PBS containing 0.05% Tween 40 (PBS-T, Merck), the bead samples were suspended in 40 μL of Laemmli buffer (2×) supplemented with 200 mM DTT and boiled at 95°C for 10 min. The samples were subjected to Western blot analyses using 4 to 8% Tris-Acetate gel (Invitrogen) either by using an anti-HA high-affinity rat MAb (3F10, Roche; 1:2000, overnight) followed by incubation with a HRP-conjugated goat anti-rat antibody (Abcam; ab97057; 1:3000, 2 h) or by using an anti-TST Ab (THE NWSHPQFEK-tagged mouse MAb, A01732, GenScript; 1:2000, overnight) followed by a HRP-conjugated polyclonal goat anti-mouse Ab (P0447, Dako; 1:3000, 2 h).
(ii) Soluble LRP6. 2 μg of purified solLRP6-Fc or solSLAM.V-Fc were mixed with 2 μL of magnetic beads (Dynabeads Protein G, Invitrogen) in 300 μL of PBS containing 0.05% Tween 40 (PBS-T, Merck) using Protein LoBind tubes (L200956G, Eppendorf) and incubated on a rotating wheel overnight at 4°C. Then, 0.5 μg of soluble HOP-TST, Hwt, HOP^A393T-TST, and Hwt^R529A-TST (or no proteins for the control experiments) were added to each bead sample and incubated on a rotating wheel for 2 h at 4°C. After five washes with PBS containing 0.05% Tween 40 (PBS-T, Merck), the bead samples were resuspended in 10 μL of Laemmli SDS reducing (4×) sample buffer (J60015, Alfa Aesar) supplemented with 200 mM DTT and were boiled at 95°C for 10 min. Finally, the samples were subjected to Western blot analyses (using 10% Bis-Tris gels [GenScript]), as described above for the membrane-anchored LRP6 coimmunoprecipitation. A HRP-conjugated, goat anti-human IgG antibody (AP113P, Merckmillipore, 1:2000, overnight) was used to reveal the Fc proteins.