Fig. 4: CgAKT phosphorylates Thr260 of CgCaspase-3/6/7 to inhibits its activation.

A Co-immunoprecipitation (co-IP) of CgCASP3 with CgAKT. HEK293T cells expressing the indicated constructs encoding mCherry-CgCasp3 and Myc-CgAkt were lysed and incubated with anti-mCherry magnetic beads overnight, Myc-tagged molecules co-IPed in this manner were resolved by SDS-PAGE and detected by immunoblotting with anti-Myc antibody. The expression of CgCASP3 and CgAKT by transfectants (INPUT) in these studies was also confirmed by immunoblot analysis. B Yeast two-hybrid assay between CgCASP3 and CgAKT. Full-length of CgCasp3 and CgAkt were fused to the pGBKT7 binding domain (BD, bait) and the pGADT7 activation domain (AD, prey), respectively, and then transformed into yeast. Shown are growth phenotypes of yeast transformants on selective media of SD/Leu-Trp- (left panel), SD/Leu-Trp-His- (central panel, interaction) and SD/Leu-Trp-His-Ade2 (right panel, interaction). C BiFC assay of CgCASP3 with CgAKT. HeLa cells were transfected BiFC plasmids expressing CgCASP3 (pBiFC-VN173-CgCasp3) only, CgAKT (pBiFC-VC155-CgAkt) only or CgCASP3 and CgAKT. Images were acquired with a confocal microscope at the EGFP channel. Bar: 10 µm. D Subcellular localization of CgCASP3 and CgAKT in HeLa cells under control and heat stress. HeLa cells were transfected mCherry-CgCasp3 and Myc-CgAkt. Images were acquired with a confocal microscope under control and heat treatment. Bar: 10 µm. E In vitro kinase activity assay of CgAKT on CgCASP3 Thr260 site. The recombinant proteins (His-CgAKT and His-CgCASP3/His-CgCASP3^T260A) were incubated in kinase buffer at 30 °C for 30 min and detected by western blotting with anti-His and anti-phosphoserine/threonine (anti-pS/pT) antibodies. F In vivo phosphorylation assay of CgAKT on CgCASP3 Thr260 site. HEK293T cells were transfected with mCherry-CgCasp3/mCherry-CgCasp3^T260A and Myc-CgAkt. The cells were lysed and incubated with anti-mCherry magnetic beads overnight, and IPed proteins were separated using SDS-PAGE gels. The phosphorylation level of CgCASP3/CgCASP3^T260A were detected by western blotting using anti-phosphoserine/threonine antibody. The cell-based transfection DEVD-ase activity assay (G.; n = 5), the CCK-8 assay for the cell viability (H.; n = 5), the lactate dehydrogenase (LDH) release assay (I.; n = 5) and the cell apoptosis rate (J.; n = 3) of HEK293T cells were transfected with mCherry-CgCasp3/mCherry-CgCasp3^T260A and Myc-CgAkt, and incubated with TNF-α + SM-164 for 8 h (n = 3). The error bars represent the S.D. Significant differences among groups were marked with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.