Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A Co-immunoprecipitation (co-IP) of Cg CASP3 with Cg AKT. 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 Cg CASP3 and Cg AKT by transfectants (INPUT) in these studies was also confirmed by immunoblot analysis. B Yeast two-hybrid assay between Cg CASP3 and Cg AKT. 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 Cg CASP3 with Cg AKT. HeLa cells were transfected BiFC plasmids expressing Cg CASP3 (pBiFC-VN173- CgCasp3 ) only, Cg AKT (pBiFC-VC155- CgAkt ) only or Cg CASP3 and Cg AKT. Images were acquired with a confocal microscope at the EGFP channel. Bar: 10 µm. D Subcellular localization of Cg CASP3 and Cg AKT 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 Cg AKT on Cg CASP3 Thr260 site. The recombinant proteins (His- Cg AKT and His- Cg CASP3/His- Cg CASP3 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 Cg AKT on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 /mCherry- CgCasp3 T260A and Myc-Cg Akt . 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 Cg CASP3/ Cg CASP3 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.

Article Snippet: The following antibodies were used: mCherry-tag (ABclonal, AE094; 1:1000 dilution), Myc-tag (ZENBIO, 390003; 1:1000 dilution), beta Actin (ABclonal, AC026; 1:1000 dilution), Cleaved-PARP1 (ZENBIO, 380374; 1:1000 dilution), Flag-tag (ZENBIO, 390002; 1:1000 dilution), HA-tag (ZENBIO, 201113; 1:1000 dilution), His-tag (ZENBIO, 230001; 1:1000 dilution), AKT1 (ABclonal, A22533; 1:1000 dilution), Phospho-AKT1-T308 (ABclonal, AP1214; 1:1000 dilution), PDPK1 (ABclonal, A1665; 1:1000 dilution), Phospho-PDPK1-S241 (ABclonal, AP0426; 1:1000 dilution), PTEN (ABclonal, A19104; 1:1000 dilution) and PIK3CA (ABclonal, A16950; 1:1000 dilution), HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF101; 1:5000 dilution) and HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF102; 1:5000 dilution).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Construct, Incubation, Magnetic Beads, SDS Page, Western Blot, Y2H Assay, Binding Assay, Activation Assay, Transformation Assay, Bimolecular Fluorescence Complementation Assay, Transfection, Microscopy, Control, In Vitro, Kinase Assay, Recombinant, In Vivo, Phospho-proteomics, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay

Journal: Communications Biology

Article Title: PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase -3/6/7 regulates heat-induced activation in oysters

doi: 10.1038/s42003-024-07184-4

Figure Lengend Snippet: A The sequence alignment surrounding T291 on oyster AKT protein. The result showed that the oyster AKT residues T291 correspond to the conserved active sites T308/T309/T305 in human AKT1/AKT2/AKT3. B The western blotting of proteins extracted from gill tissues of C. gigas and C. angulata during heat stress with PI3K-AKT pathway’s antibodies. Gi and An represent the C. gigas and C. angulata under control condition. HGi and HAn represent the C. gigas and C. angulata under heat stress. C In vivo phosphorylation assay of different Cg AKT mutants on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A /Myc- CgAkt T291DS464D . 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 Cg CASP3 were detected by western blotting using anti-phosphoserine/threonine antibody. D In vitro kinase activity assay of Cg AKT/ Cg AKT T291AS464A on Cg CASP3 Thr260 site. The recombinant proteins (His- Cg AKT/His- Cg AKT T291AS464A /His- Cg AKT T291DS464D and His- Cg CASP3) were incubated in kinase buffer at 30 °C for 30 min and detected by western blotting with anti-His and anti-phosphoserine/threonine antibodies. The western blotting ( E ), The cell-based transfection DEVD-ase activity assay ( F .; n = 5), the CCK-8 assay for the cell viability ( G .; n = 5), the lactate dehydrogenase (LDH) release assay ( H .; n = 5) and the cell apoptosis rate ( I .; n = 3) of HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A /Myc- CgAkt T291DS464D , and incubated with TNF-α + SM-164 for 8 h. J In vivo phosphorylation assay of Cg AKT and its upstream regulators in PI3K-AKT pathway ( Cg PDPK1, Cg PIK3CA and Cg PTEN) on Cg CASP3 Thr260 site. HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A , His- CgPdpk1 , HA- CgPik3ca and Flag- CgPten . 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 Cg CASP3 were detected by western blotting using anti-phosphoserine/threonine antibody. K The western blotting of HEK293T cells were transfected with mCherry- CgCasp3 and Myc- CgAkt /Myc- CgAkt T291AS464A , His- CgPdpk1 , HA- CgPik3ca and Flag- CgPten , and incubated with TNF-α + SM-164 for 8 h. 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.

Article Snippet: The following antibodies were used: mCherry-tag (ABclonal, AE094; 1:1000 dilution), Myc-tag (ZENBIO, 390003; 1:1000 dilution), beta Actin (ABclonal, AC026; 1:1000 dilution), Cleaved-PARP1 (ZENBIO, 380374; 1:1000 dilution), Flag-tag (ZENBIO, 390002; 1:1000 dilution), HA-tag (ZENBIO, 201113; 1:1000 dilution), His-tag (ZENBIO, 230001; 1:1000 dilution), AKT1 (ABclonal, A22533; 1:1000 dilution), Phospho-AKT1-T308 (ABclonal, AP1214; 1:1000 dilution), PDPK1 (ABclonal, A1665; 1:1000 dilution), Phospho-PDPK1-S241 (ABclonal, AP0426; 1:1000 dilution), PTEN (ABclonal, A19104; 1:1000 dilution) and PIK3CA (ABclonal, A16950; 1:1000 dilution), HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF101; 1:5000 dilution) and HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF102; 1:5000 dilution).

Techniques: Sequencing, Western Blot, Control, In Vivo, Phospho-proteomics, Transfection, Incubation, Magnetic Beads, SDS Page, In Vitro, Kinase Assay, Recombinant, Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay