NADH restrained translation by enhancing the binding of LDHA and eEF2. (A) Coimmunoprecipitation (IP) of DAMI whole-cell lysates with anti-LDHA antibody and ribosomal protein antibodies. (B) Co-IP of Flag-LDHA and HA-eEF2 in HEK-293T treated with pyruvate, lactate, and NAD⁺. Negative control (NC) was transfected with empty HA/Flag-tag plasmid and used for nonspecific binding control. (C) Co-IP of Flag-LDHA and HA-eEF2 in HEK-293T treated with different concentrations of NADH; the gray values of eEF2-HA IP bands were counted (n = 3). (D) The protein expression of eEF2 and LDHA in mouse or rabbit reticulocyte lysate was detected by using western blot. β-Actin was used as a loading control. (E) The levels of protein synthesis were measured when different concentrations of LDHA proteins were added in the rabbit reticulocyte translation system (n = 3). (F) The levels of protein synthesis were measured when different concentrations of NADH were added in the rabbit reticulocyte translation system with or without 5 μg LDHA (n = 3). (G) The levels of protein synthesis were measured when different concentrations of NAD⁺ were added in the rabbit reticulocyte translation system with 5 μg LDHA (n = 3). (H) The levels of protein synthesis were measured when glucose (5 mM), pyruvate (1 mM), and lactate (2 mM) were added in the rabbit reticulocyte translation system with 5 μg LDHA (n = 3). (I) Megakaryocyte PPF assay in LdhA^f/f and LdhA^−/− mice liver fetal MKs incubated with 1 mM NADH. Statistical analysis of the PPF-MK/total MK ratio is shown (n = 4). Scale bar, 100 μm. *P < .05, **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; RPL28, ribosomal protein L28; RPL35, ribosomal protein L35; RPS3A, ribosomal protein S3A.