Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: Lysine-222 succinylation reduces lysosomal degradation of lactate dehydrogenase a and is increased in gastric cancer
Figure Lengend Snippet: CPT1A binds to and succinylates LDHA at K222. a and b KAT2A does not bind to LDHA or its mutant K222E. AGS cells were transfected with Flag-KAT2A in combination with HA-LDHA or HA-K222E plasmid. KAT2A fusion protein was pulled down by Flag antibody, LDHA or K222E was detected using HA antibody. c - e CPT1A interacts with LDHA and its mutant K222E in AGS and 293 T cells. Cells were transfected with Flag-CPT1A in combination with HA-LDHA ( c , d ) or HA-K222E ( e ). The co-IP assay was performed similarly as above ( a , b ). f and g CPT1A promotes the succinylation of LDHA ( f ), but not of its mutant K222E ( g ). AGS cells were transfected with the indicated plasmids. Then, HA-LDHA was immunoprecipitated and its succinylation analyzed using either pan-succinylation or K222suc-specific antibody. h and i Knockdown efficiency of three CPT1A shRNAs in AGS cells was determined by western blot ( h ). CPT1A deficiency decreased the succinylation of LDHA ( i )
Article Snippet: CPT1A and KAT2A cDNA plasmids were purchased from Sino Biological (Beijing, China) and subsequently cloned into the pRF-FLAG vector.
Techniques: Mutagenesis, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot