Protein Expression Analysis in Cultured Cells

Total cell lysates and nuclear protein-enriched lysates from cultured cells or ectopic bone implants were obtained using the appropriate cell lysis buffers, and Western blot analysis was performed as described previously.^{10 ,12 (link)} Briefly, proteins were separated by SDS-­PAGE, and protein-containing nitrocellulose membranes were incubated overnight with primary antibodies specific for the following proteins: AMPK (#2532; Cell Signaling Technology), p-AMPK^T172 (#2535, Cell Signaling Technology), ATF4 (#11815, Cell Signaling Technology), β-­actin (A5441, Sigma-Aldrich), eIF2α (#9722, Cell Signaling Technology), p-eIF2α^S51 (#9721, Cell Signaling Technology), Lamin A/C (sc-376248, Santa Cruz Biotechnologies), PHGDH (#66350, Cell Signaling Technology) and PSAT1 (NBP1-55368, Bio-Techne). Appropriate HRP-conjugated secondary antibodies were used for chemiluminescent detection of proteins (Western Lightning Plus, PerkinElmer).