Anti lactyl lysine

Isolated samples were decalcified and embedded for sectioning into 5 μm. Sections were deparaffinized and retrieved with antigen with 0.25% trypsin (Solarbio) for 30 min at 37 °C. Subsequently, 3% hydrogen peroxide was added for 15 min, and 5% normal goat serum (Solarbio) was added for block for 1 h at room temperature. The antibodies anti-LDHA (Abcam) and anti-lactyl-lysine (PTM Bio) were added to the sections overnight at 4 °C. The second antibody was added and then counterstained with hematoxylin (Solarbio). Notably, the distal side of the upper and middle third of mesiobuccal roots was considered the tension zone due to the tipping movement of the teeth. The quantification was performed using ImageJ software.

Total protein was extracted and lysed in RIPA reagent (Solarbio) with 1% PMSF (Solarbio). Specifically, the nuclear protein was extracted using the Nuclear Protein Extraction Kit (Solarbio). Then, the lysates were separated on 10% or 15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) following transfer to a polyvinylidene fluoride membrane (PVDF; Millipore). After blocking with 5% skimmed milk, these membranes were incubated with the following primary antibodies: anti-RUNX2 (Cell Signaling Technology), anti-ALP (Proteintech Group, Inc.), anti-LDHA (Abcam), anti-β actin (Abcam), anti-Histone 3 (Abcam), and anti-lactyl-lysine (PTM Bio) overnight at 4 °C. Following washing with Tris-buffered saline containing 0.05% Tween 20, the second antibody was added and incubated for 1 h. The visualization was performed with the ECL chromogenic substrate (Millipore).