Biotinylation and LRP6 Pulldown in HEK293 Cells

MAMDC4 was overexpressed in HEK293 cells in 10cm plates as described above. When cultures reached 90% confluence plates were transferred to room temperature (RT), washed 2 X with PBS and complete media with 0.5mg/ml Sulfo-NHS-SS-Biotin was added (cat. 21331; ThermoFisher). Cells were incubated 30min at RT, washed with 50mM Tris pH8.0 and 4 X with cold PBS. Cells were lysed in NP-40 lysis buffer [10 (link)] and 950ug of total protein in equal concentration was used for each pull down. Lysates were incubated with NeutrAvidin Agarose Resin (cat. 29200; ThermoFisher) for 1hr at 4°C and washed 4 X with lysis buffer. Samples were resuspended in 30ul of sample buffer, and immunoblot was probed with antibodies for LRP6 (cat. 2560; CST). Immunoblots were imaged using a Licor-Odyssey infrared imager. LRP6 pull down was normalized to input levels.