Rabbit anti lrp6 c5c7

Primary antibodies: rabbit anti-AP4 (TFAP4) serum (1:2000, a gift from Takeshi Egawa [5 (link)]); rabbit anti-LRP6 (C5C7) (1:500, Cell Signaling Technologies Cat. # 2560); mouse anti-ACTIN (clone C4) (1:500, EMD Millipore Cat. # MAB1501).
Secondary antibodies: peroxidase AffiniPure goat anti-rabbit IgG (H+L) (1:7500, Jackson ImmunoResearch Laboratories Cat. # 111-035-003); IRDye 800CW donkey anti-rabbit IgG (H+L) (1:10,000, Li-Cor Cat. # 925–32213); IRDye 800CW donkey anti-mouse IgG (H+L) (1:10,000, Li-Cor Cat. # 926–32212).
Primary and secondary antibodies used for detection with the Li-Cor Odyssey imaging system were diluted in a 1 to 1 mixture of Odyssey Blocking Buffer (Li-Cor Cat. # 927–40000) and TBST (Tris buffered saline (TBS) + 0.1% Tween-20), and those used for detection by chemiluminescence were diluted in TBST + 5% skim milk. Primary antibody incubations were done overnight at 4°C, and secondary antibody incubations were done for 1 hr at room temperature (RT).

Protein samples dissolved in sample loading dye were separated by SDS on 8% acrylamide‐bisacrylamide gel and transferred to polyvinylidene fluoride membrane (GE Healthcare Life science, Little Chalfont, UK). Blots were blocked in 5% milk powder for 1 h at room temperature (RT) and incubated with the following antibodies: rabbit anti‐GFP ((D5.1) XP^®, 1 : 1000; Cell Signaling Technology) and rabbit anti‐Lrp6 (C5C7, 1 : 1000; Cell Signaling Technology, Danvers, MA, USA), ECL Rabbit IgG (HRP‐linked F(ab)2 fragment from donkey, 1 : 2500; GE Healthcare Bio‐Sciences), rabbit anti‐TfR2 (1 : 2000; Abcam) and mouse anti‐Caveolin1 (1 : 2000; BD Transduction Laboratories, Franklin Lakes, NJ, USA), mouse anti‐β‐ catenin (1 : 2500; BD Biosciences, Franklin Lakes, NJ, USA). Cellular localization kit (Cell Signaling) was used for Histon 3B and β‐tubulin (1 : 1000). Blots are quantified with imagej (ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA) plugin (Analyze→Gels).