Western blotting of cell lysates.

Primary mammary epithelial cells were isolated from chopped mammary glands as previously described and then directly lysed for protein analysis (7). For analysis of cultured cells, cells were solubilized in either standard lysis buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 50 mM glycerophosphate, 0.5% Triton X-100) or radioimmunoprecipitation assay (RIPA) lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate) with freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific). Western blotting of SDS-PAGE gels was performed as described previously, using the following primary antibodies from Cell Signaling Technology: anti-Lrp5 (5731), anti-Lrp6 (3395), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (2118), anti-p-Lrp6 (2568), anti-p-AKT (T308) (2965), anti-p-AKT (S473) (4058), anti-AKT (4685), anti-p-p70 S6 kinase (T389) (9205), anti-p70 S6 kinase (2708), anti-p-4E-BP1 (T37/46) (2855), anti-4E-BP1 (9452), anti-p-S6 ribosomal protein (S235/236) (2211), anti-S6 ribosomal protein (2217), anti-p-SAPK/JNK (T183/Y185) (4668), anti-SAPK/JNK (9252), anti-p-p38 (T180/Y182) (9215), anti-p38 (9212), anti-p-ERK1/2 (T202/Y204) (4377), anti-ERK1/2 (4695), anti-p-LKB1 (S428) (3482), anti-LKB1 (3047), anti-p-AMPKα (T172) (2535), and anti-AMPKα (2603). Anti-β-actin antibody was purchased from Sigma (A5441), and antivinculin antibody was purchased from Millipore (95-386).