Wnt3a

The implants attached with primary osteoblasts were washed with ice-cold PBS and lysed to release the whole proteins by RIPA buffer with 1 mM PMSF. The cell lysates were agitated at 4 °C for 30 min and then centrifuged at 4 °C for 20 min. The protein concentration was determined by the BCA assay. The protein extracts (30 μg per sample) were separated by 8% or 10% Tris-glycine SDS-PAGE and then transferred onto PVDF membranes (Millipore) after mixed with 5× loading buffer. The PVDF membranes were blocked in TBST (Tris Buffer Saline, 0.5% Tween-20) containing 5% BSA for 1 h and incubated overnight at 4 °C with primary antibodies to OCN (1:1000, Abcam, Cambridge, MA, USA), Runx2 (1:1000, Biorbyt Ltd., Cambridge, UK), Wnt3a (1:1000, Novus Biologicals, Littleton, CO, USA), Lrp6 (1:1000, Lifespan Bioscience, Seattle, WA, USA), β-catenin (1:1000, Millipore, Billerica, MA, USA), β-Tubulin (1:3000, Bioworld technology, Inc., Louis Park, MN, USA), and β-Actin (1:3000, Bioworld) in TBST containing 5% BSA. The membranes were then incubated with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at RT, and visualized by an ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). Semi-quantitative analysis was performed using the QuantityOne Software (Bio-Rad). β-Actin or β-Tubulin was used as an internal control for normalization.

The procedures of protein extraction and western blotting were described as in details in our previous study^{29 (link)}. In brief, cells in the PEMF group were exposed to PEMF at 37 °C for 3 days (2 h/day), and then protein lysates were prepared were prepared using RIPA buffer supplemented with 1 mM PMSF on ice. After quantification, protein extracts in equal amounts were loaded on a 10% Tris–glycine SDS-PAGE gel, and then electrotransferred to PVDF membranes. The blots were blocked in 5% bovine serum albumin (BSA) for 1 h, followed by incubation with primary antibodies against OCN (Abcam, Cambridge, MA), Runx2 (Biorbyt Ltd., Cambridge, UK), Wnt3a (Novus Biologicals, Littleton, CO), p-GSK-3β (Abcam), β-catenin (EMD Millipore, Billerica, MA) and β-tubulin (Bioworld technology, Inc., Louis Park, MN) in TBST containing 5% BSA overnight at 4 °C. β-tubulin was employed as the protein loading control. After incubation with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at room temperature, the membranes were visualized using the ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). The Quantity One Software (Bio-Rad) was used to quantify the densities of the bands.

Immunoblotting was performed as described previously (6, 24 (link)). Blots were probed with Streptavidin-HRP (Cell Signaling Technology #3999; RRID:AB_10830897) or antibodies used according to manufacturer's recommendations: WNT4 (R&D Systems, MAB4751; RRID:AB_2215448); WNT3A (Novus Biologicals, MAB13242); anti-HA-HRP conjugate (Cell Signaling Technology #2999; RRID:AB_1264166); DHRS2 (Sigma HPA053915; RRID:AB_2682307); mTOR (Cell Signaling Technology #2983; RRID:AB_2105622); STAT1 (Sigma HPA000931; RRID:AB_1080100).; MCL1 (Cell Signaling Technology #5453; RRID:AB_10694494); ACC1 (ACACA, Abcam ab45174; RRID:AB_867475); ACC1-pS79 (Cell Signaling Technology #3661; RRID:AB_330337); AMPKα (Cell Signaling Technology #2532; RRID:AB_330331); AMPKα-pT172 (Cell Signaling Technology #2535; RRID:AB_331250). We note that recent lots of WNT4 MAB4751 show substantially increased non-specific background relative to our prior studies (6, 24 (link)), and can detect overexpressed WNT4 but no longer reliably detect endogenous WNT4 protein.