Anti l lactyl lysine

Individual embryos were injected at HH4 with LDHA/B MO mix (0.75 mM each LDHA and LDHB) on one half and FITC-Control MO (0.75 mM) on the other half as previously stated. A non-fluorescent construct (pTK-Luciferase) was used as carrier DNA. The embryos were allowed to develop until HH9 at which point they were collected in Ringer’s solution and half of the head was dissected and assigned to control or treated samples The samples were subjected to the cell suspension staining protocol described above while being protected from light (n = 12 half embryonic heads for both FITC-control and LDHA/B MO samples). The antibodies used for this flow cytometry experiment are as follows: rabbit polyclonal anti-L-Lactyl-lysine (PTM Biolabs, PTM-1401 polyclonal) (1:100) and Alexa Fluor 647-conjugated AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, 711-607-003) (1:1000). The samples were analyzed on a BD FACSCelesta flow cytometer, measuring Alexa647 and FITC fluorescence intensity of each sample.

Total proteins in cells were extracted using RIPA buffer (Aspen Biological, Wuhan, China) and protease inhibitors (MedChemExpress, SNJ, USA). Protein concentration was measured using the BCA kit (Aspen). Equal amounts of protein were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with milk at room temperature for 1 h. PVDF membranes were washed three times with TBST for 10 min each, and then incubated overnight at 4 °C with the appropriate primary antibody. Following another round of washing, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were developed using ChemiDoc XRS gel imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this experiment are listed below: Anti-beta Tubulin (PTM-6414, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys18) (PTM-1427RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys14) (PTM-1414RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys9) (PTM-1419RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl Lysine (PTM-1425, PTMBIO, Zhejiang, China); Anti-Histone H3 (PTM-6621, PTMBIO, Zhejiang, China); SLC25A29 (26663-1-AP, Proteintech, Wuhan, China).