Fig. 5. TOFA reduces tubulin palmitoylation both in interphase and mitosis.

Tubulin palmitoylation was reduced during mitosis and further decreased by acute TOFA treatment. a The PLA between α-tubulin and click-labeled palmitate (palmitate-488) was performed using CGL2 cells. The representative images show the control cell (No PA) and the PLA signal between α-tubulin and palmitate (palmitate-α-tubulin PLA) in interphase and mitotic cells that were untreated (−) or treated with TOFA as indicated. The insets below show magnified views of regions with colocalization between PLA signals and palmitate-488. b Relative intensity of palmitate-α-tubulin PLA in a single cell was measured. The scatter plot shows the interquartile distribution of the PLA intensity in the group of cells described in (a). The numbers above indicate the numbers of cells assayed from two independent experiments. *P < 0.05 by Mann–Whitney rank-sum test. c Illustration of the acyl-biotin exchange assay. d Palmitoylated proteins in MDA-MB-231 cells were purified using acyl-biotin exchange assay and then subjected to western blotting for α-tubulin detection. HAM addition (+) is required for palmitate cleavage, biotinylation, and subsequent purification of the palmitoylated proteins, and the omission of HAM (−) served as the negative control. Input lanes show equal amounts of total tubulin in the cell lysates were applied in each reaction. The western blots show the levels of total cellular tubulin (Input) and palmitoylated fractions of tubulin purified by streptavidin agarose (SA-IP) from total cell lysates (Input) of untreated (−) and TOFA-treated cycling and enriched mitotic cells. The input tubulin was first normalized with PCNA to adjust loading variations and then served to normalize the purified tubulin. The values below each band indicate the level of purified α-tubulin after normalizing to input tubulin, relative to untreated cycling cells; mean ± SD from three independent experiments is shown.