Containingprotease and phosphatase inhibitors

Details for the FGF23 signaling
assay were described previously.^{30 (link)} Briefly,
HEK293 cells were transfected with empty vector control (Ctrl) or
plasmids expressing either KLWT or KLΔ9. Twenty-four hours after
transfection, cells were incubated in serum-free medium for 2 h and
then either 50 ng/mL of bFGF (basic fibroblast growth factor) or 10
ng/mL of FGF23 (R&D Systems, Minneapolis, MN) was added to the
wells. The cells were incubated for 15 min at 37 °C and then
were immediately washed in PBS and lysed in RIPA buffer containing
protease and phosphatase inhibitors (Roche, Mannheim, Germany). Various
incubation times and FGF23 concentrations were used as indicated in
the p-ERK phosphorylation kinetics and FGF23 dose–response
curve experiments. After lysis, samples were prepared for SDS-PAGE
as described previously.^{6 (link)}

HEK 293T cells
were transiently
transfected with plasmids encoding wild-type S1p2 and S1p2 R150H using
Attractene (Qiagen). Twenty-four hours after transfection, cells were
serum-starved overnight and then stimulated for 2–90 min using
0.5–1 μM S1P. Lysis was performed using a sodium dodecyl
sulfate (SDS)-based buffer [2% SDS and 50 mM Tris (pH 6.8)] containing
protease and phosphatase inhibitors (Roche). Equal amounts of protein
were separated using NuPage 4 to 12% Bis-Tris gels (Life Technologies)
and transferred to nitrocellulose membranes (Bio-Rad Laboratories).
After being blocked in 5% nonfat milk in TBS containing 0.05% Tween
20, blots were incubated with primary antibodies overnight. Secondary
antibodies that were used contain labels for near-infrared detection
with a LI-COR Odyssey SA system. Signals were quantitated using LI-COR
Studio-Lite version 3.1.