Mice
Cell lines
Analysis of bone phenotype
Plasmids
Antibodies
Cellular fractionation
Immunofluorescence staining
Coimmunoprecipitation to analyze association with p300
Primer sequences used for PCR
Luciferase reporter assay
RNA interference
Biochemical analysis of osteocytes in vivo and in vitro
Measurement of intracellular ROS level
FSS experiments
ChIP assay
Hemilateral hindlimb unloading experiments
Statistical analysis
Total RNA was isolated using an RNA extraction kit (RNeasy, Qiagen or OMEGA Bio-Tech), and complementary DNA (cDNA), obtained using the First Strand cDNA Synthesis Kit (Qiagen or Fermentas), was amplified by quantitative polymerase chain reaction (PCR) using either QuantiTect SYBR Green PCR Master Mix (Qiagen) with a 7300 Real-Time PCR system (Applied Biosystems) or SsoFast EvaGreen Supermix with a CFX96 C1000 thermal cycler (Bio-Rad). All reactions were run in triplicate. The mRNA expression levels of individual genes were normalized to GAPDH or EF-1α (mouse) or β-actin (human) expression.