Changes in Acetyl CoA Levels during the Early Embryonic Development of Xenopus laevis
Figure 4
Whole-embryo acetylation levels of a number of proteins in Xenopus embryos appear to correlate with the pattern of change in acetyl CoA levels.
(A) 20 embryos collected at different stages of development were lysed in 80 µl lysis buffer and approximately 40 µg of soluble protein was separated by SDS-PAGE, transferred to a PVDF membrane and probed with anti-acetyl lysine antibody. Equal protein loading was checked by Ponceau staining of the membrane and a Coomassie stain of a gel run in parallel. A section of the Ponceau stained membrane is shown (the complete Ponceau stained membrane and the Coomassie stained gel are shown in Figure S3). Changes in the abundance of a number of acetylated proteins, including those with molecular weights of approximately 230 KDa, 100 KDa, 16 KDa, and 18 KDa (indicated by arrows), appear to follow the overall pattern of change in acetyl CoA levels. (B–D, top graphs) Embryos at different stages of development were collected and Western blotting, with anti-acetyl lysine antibody, was performed as above. The intensity/size of acetylated protein bands at approximately 230 KDa (B), 100 KDa (C) and 16–18 KDa (D) were quantified, normalised to stage 4, and plotted against different stages of development together with levels of acetyl CoA (taken from Figure 3 and expressed relative to stage 4). Band intensity/size was normalised against stage 4 as this stage is included in all the blots performed. Each data point represents a single measurement or the mean +/− SEM of 2–7 measurements. The number of independent measurements (using samples from independent fertilisations) performed for each stage was as follows: S1 (4); S2 (4); S3 (1); S4 (7); S8/9 (1); S9/10 (7); S12.5/13 (4); S20 (5); S21 (2); S27/28 (4); S39/39 (2); S40 (3). (B–D, bottom graphs) The means of normalised band intensity/size for acetylated proteins at approximately 230 KDa (B), 100 KDa (C) and 16–18 KDa (D) were plotted against acetyl CoA levels (taken from Figure 3 and also normalised to stage 4) and R2 values are shown.
