Fig. 7
Effect of Protein Tyrosine Phosphorylation on Protein Palmitoylation and Interplay between Protein Palmitoylation and Calcium. (A) Effect of 2BP on intracellular calcium ([Ca2+]i) levels in sperm. Sperm loaded with the Fluo4-AM fluorescence probe were treated with 2BP, and fluorescence intensity was dynamically monitored. Data were normalized by setting initial raw intensity values to 1.00 in the blank group (n = 7 repeats, one individual mouse per repeat). The corresponding significant P-values are listed in (B). (C) Effect of PKA/PKC inhibitors and calcium chelators on protein palmitoylation in sperm. Sperm were treated with 30 µM H89, 30 µM CC, 20 µM BAPTA-AM, and 3 mM EGTA, respectively. Protein palmitoylation was detected using the ABE method. The loading controls β-tubulin were confirmed by Western blotting. The level of protein palmitoylation was quantified using ImageJ software (D). The gray intensities of the bands indicated by arrows in (C) were normalized against the total protein amount loaded. Data are presented as means ± SEM (n = 3 or 4 repeats, pooled samples from 12 mice per repeat), with the DMSO group serving as the vehicle control. A P-value < 0.05 denotes statistical significance