Ab119796

For femur samples, the preparation process was performed by Beijing Genepool Biotechnology Company Limited. Briefly, tissue samples were fixed for 2 days with 4% paraformaldehyde and dehydrated according to standard protocols. Paraffin embedded sections with 5 mm thick were placed in a 65 °C oven for 2 h, dewaxed to water, and washed with PBS for three times for 5 min each time. The slices were placed in EDTA buffer for microwave repair and boiled for 10min. The sections were blocked with 5% normal goat blocking serum for 20 min at room temperature, and then incubated with the primary antibodies anti-PCK2 (6924S, Cell signaling technology, 1:250) and anti-PFKP (ab119796, Abcam, 1：250) overnight at 4 °C. Washed with PBS for 3 times, 5min each time, the secondary antibodies corresponding to the species of the primary antibody were added to cover the slices in the circle, and incubated at room temperature in dark for 60 min. The nuclei were stained by DAPI for 60 min. After the slices were slightly dried, they were sealed with anti-fluorescence quenching sealing agent. Then the images were scanned and analyzed. Immunohistochemical (IHC) staining was also used for validation of PFKP expression, the antibody was ab119796 (anti-PFKP, Abcam, 1:100, Cambridge, UK).

Cell pellets were extracted from GelMA as described above. The pellets were lysed in RIPA lysis buffer (medium) with cocktail (Huaxingbochuang) and phosphatase inhibitors (Huaxingbochuang), and the protein concentration was determined using the BCA kit. The Western blot was performed as previously described [24 ]. The following antibodies were used: anti-GAPDH (ab9485, Abcam), anti-PCK2 (6924S, Cell signaling technology), anti-PFKP (ab119796, Abcam), anti-PFKM (ab154804, Abcam), anti-PFKL (ab154804, Abcam), anti-FAK (ab40794, Abcam), anti-vinculin (13901, Cell signaling technology), anti- Runt-related transcription factor 2 (RUNX2) (ab12556, Abcam), anti-phospho-AKT S473 (4060; Cell Signaling Technology, Boston, MA, USA), anti-AKT (4691, Cell Signaling Technology), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370, Cell Signaling Technology), anti-p44/42 MAPK (Erk1/2) (4695, Cell Signaling Technology).