Fig. 6: ME1 dimerization and ME1-mediated flux are suppressed, whereas the fluxes through MDH1 and PCK1 are elevated in Q cells.
a Immunoblots and protein quantification of ME1, MDH1, and PCK1 in P and Q MEFs. Vinculin was used as a loading control. Values are the mean ± SEM of 4 biologically independent experiments. ns P = 0.1412, ****P < 0.0001 using unpaired two-tailed t-test. ME1 malic enzyme 1, MDH1 malate dehydrogenase 1, PCK1 phosphoenolpyruvate carboxykinase 1. b Schematic of ME1 acetylation by ACAT1 and deacetylation by SIRT6. c BS3 cross-linking assay in P and Q MEFs. After the addition of BS3, shifted bands that indicated protein–protein cross-linking could be seen; the shifted bands are marked with an asterisk. Representative data from six independent experiments are presented. d Coimmunoprecipitation (Co-IP) assay shows that ME1 dimerization is enhanced in P cells. Both Flag-tagged ME1 and Myc-tagged ME1 were expressed in MEFs, and the extracts were subjected to Co-IP with an anti-Flag tag antibody. Both immunoprecipitates with Flag (IP-Flag) and whole-cell lysates (WCL) were analyzed by Western blotting and protein quantification with means ± SEMs (n = 3–4) are shown. P value by unpaired two-tailed student’s t-test is indicated in the figure except for ****P < 0.0001 and ns (SIRT P = 0.4538, Myc-ME1 P = 0. 3851, Flag-ME1 P = 0.3780). e Co-IP assay shows that ME1 acetylation is enhanced in P cells. Extracts were subjected to Co-IP with the anti-ME1 antibody. Both immunoprecipitates with endogenous ME1 (IP-ME1) and whole-cell lysates (WCL) were analyzed by Western blotting and protein quantification with means ± SEMs (n = 3–4) are shown. ****P < 0.0001 and ns (SIRT P = 0.9095, ME1 P = 0.8684) using unpaired two-tailed t-test. f Schematic of ME1 acetylation and dimerization in P and Q MEFs. g Comparison of the quantified ME1-, MDH1-, and PCK1-mediated fluxes (estimated flux ± SD) in P and Q MEFs. P cells are shown in black bars, and Q cells are shown in gray bars. All flux fits passed the chi-square goodness-of-fit test. P values by unpaired two-tailed student’s t-test are indicated except for ****P < 0.0001. h Schematic of ME1-, MDH1- and PCK1-mediated fluxes in P and Q MEFs.
