Western blots were performed using anti-actin antibody (clone C4; Millipore, Temecula, CA), mouse RYK antibody (R&D Systems), active β-catenin (Cell Signaling Technology, Danvers, MA) and β-catenin (Bethyl, Montgomery, TX), visualized with ECL (SuperSignal West Femto, Thermo Fisher Scientific) and quantified by LAS3000 software.
For RT-PCR analysis, RNA was purified from mMSCs and converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific) and amplified using specific primers for mRyk (5′-ATC CTA CCT TGC GGA TGA AG-3′, 5′-CAT CAT CGT CAC CTG AAC TT-3′), Ryk-ICD (5′-TCC AAG GTA CTT TTG GGC G-3′, 5′-TGT CCC CTA GGC AGT GGT AG-3′) and Ryk-ECD (5′-CAG TCA CTA CGC TCT GTC CT-3′, 5′-GCT CGA CCC GAA ACA CTG AT-3′). Real-time quantitative PCR (RQ-PCR) was performed with the Rotor-Gene 6000 system (Corbett Life Science, Australia) and SYBR Premix Ex Taq (TaKaRa, Japan). Relative levels of PCR products were determined after normalization to an endogenous Gapdh control.
For RT-PCR analysis, RNA was purified from mMSCs and converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific) and amplified using specific primers for mRyk (5′-ATC CTA CCT TGC GGA TGA AG-3′, 5′-CAT CAT CGT CAC CTG AAC TT-3′), Ryk-ICD (5′-TCC AAG GTA CTT TTG GGC G-3′, 5′-TGT CCC CTA GGC AGT GGT AG-3′) and Ryk-ECD (5′-CAG TCA CTA CGC TCT GTC CT-3′, 5′-GCT CGA CCC GAA ACA CTG AT-3′). Real-time quantitative PCR (RQ-PCR) was performed with the Rotor-Gene 6000 system (Corbett Life Science, Australia) and SYBR Premix Ex Taq (TaKaRa, Japan). Relative levels of PCR products were determined after normalization to an endogenous Gapdh control.