Phospho akt thr308

Chemical agents employed in the studies include the pan-PI3K inhibitor PI-103 (Cayman Chemical), Akt inhibitor VIII (Akti; Millipore), and the diC8 phosphoinositides PI(3,4,5)P₃, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from Echelon Biosciences. The antibodies used targeted GFP (Santa Cruz sc-9996), phospho-Ser/Thr Akt substrate sites (Cell Signaling 9614), Akt phospho-Thr308 (Cell Signaling 9275), or HCN2 (Abcam ab19346). Active recombinant Akt1 protein was from Millipore.

The Btk inhibitors ibrutinib and PLS-123 were synthesized at the laboratory of Dr. Zhengying Pan at Peking University Shenzhen Graduate School according to a previously published procedure [14 (link), 38 (link)]. Antibodies against Caspase-3 (#9662), Caspase-8 (#9746), Caspase-9 (#9508), XIAP (#2045), BCL-xL (#2764), BCL-2 (#2870), MCL-1 (#5453), BAX (#5023), phospho-Tyr223-Btk (#5082), Btk (#8547), phospho-Tyr759-PLCγ2 (#3874), phospho-Tyr1217-PLCγ2 (#3871), PLCγ2 (#3872), phospho-p38 (#9211), p38 (#8690), phospho-Thr308-AKT (#9275), AKT (#9272), phospho-Ser2448-mTOR (#2971), mTOR (#2972), phospho-ERK1/2 (#4370) and ERK1/2(#9102) were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PARP, phospho-Tyr551-Btk, β-actin (A5441), IgM, Ki-67 antibodies were obtained from BD Biosciences, Sigma (St. Louis, MO, USA) and Abcam (Cambridge, MA).

Antibodies against the following proteins were used in this study: TMBIM6/BI-1 (1:100, ab51905), and RPL19 (1:1000, ab128648) (all from Abcam, Cambridge, UK); RICTOR (1:2000, A300-459A) (Bethyl Laboratories, Montgomery, TX, USA); AKT (1:1000, #9272), GST (1:1000, #2622), mTOR (1:1000, #2972), mTOR (1:1000, #4517), NDRG1 (1:1000, #9408), phospho-Ser473-AKT (1:1000, #9271), phospho-Ser939-TSC2 (1:1000, #3615), phospho-Thr308-AKT (1:1000, #4056), TSC2 (1:1000, #4308), phospho-Thr346-NDRG1 (1:1000, #3217), SIN1 (1:1000, #12860), p70 S6 Kinase (1:100, #9202), RICTOR (1:1000, #2114), RICTOR (Sepharose bead conjugate, #5379), and RAPTOR (1:1000, #2280) (all from Cell Signaling Technology, Danvers, MA, USA); HA (1:2000, 11867423001) (Roche Diagnostics, Basel, Switzerland); actin (1:1000, sc-47778), Ki-67 (1:100, sc-15402), phospho-Thr450-AKT (1:1000, sc-293094), RPL19 (1:1000, sc-100830), and RPS16 (1:1000, sc-102087), (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies (1:10,000) for immunoblotting (Jackson ImmunoResearch, West Grove, PA, USA) and immunoprecipitation (1:5000, sc-2006) (Santa Cruz Biotechnology) were also used. Insulin (I3769), EGF (E9644), and IGF1 (I3769) were from Sigma-Aldrich. BAPTA-AM (B6769), BAPTA (B1212), and EGTA-AM (E1219) were from Thermo Fisher Scientific (Waltham, MA, USA).

Antibodies to mTOR, rictor, raptor, mLST8, p18, phosphoThr1135 rictor, phospho-Ser240/244 S6 ribosomal protein, total S6 ribosomal protein, phospho-Thr389 p70 S6 kinase 1, phospho-Ser65 and phospho-Thr37/46 4E-BP1, p-Ser757 ULK1, phospho-Ser473 Akt, phospho-Thr308 Akt and GAPDH were from Cell Signaling Technology. Monoclonal anti-FLAGM2-Peroxidase (HRP) antibody, Flag M2 antibody, anti-FLAGM2 Affinity Gel, anti-HA and dimethyl pimelimidate were from Sigma Aldrich; PP6 subunit antibodies (SAPS1, SAPS2, SAPS3 and ANKRD28) from Bethyl laboratories; antibodies to ATP6V1B2, ATP6V0A2 and LAMP2 from Abcam; antibody to ATP6V1A was from GeneTex; polyethylenimine (PEI) from Polysciences; Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence Western blotting kit were from Amersham Bioscience; [³²P-γ]ATP was from Perkin Elmer; Protein G-Sepharose and immobilized glutathione from Pierce; Precast NuPAGE polyacrylamide Bis-Tris gels, Colloidal Coomassie, LDS sample buffer and Dextran Oregon Green 514 (D-7176) were from Invitrogen; dialyzed Fetal Bovine Serum (Cat # 26400036) was from ThermoFisher Scientific; sequencing-grade trypsin and CellTiter-Glo was from Promega; and microcystin-LR was purchased from Dr Linda Lawton (Robert Gordon University, Aberdeen, UK).

CLVM EC, HUVEC, and HDLEC were expanded in EGM-2 supplemented with 20% FBS. Cells were washed with PBS and lysed using RIPA buffer (Teknova) with phosphatase and protease inhibitor cocktail (Roche). Cell lysates were separated by electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were probed with the following primary antibodies: VEGFR-2 (1:1000, Cell Signaling Technology), PROX1 (1:200, R&D Systems), VEGFR-3 (1:250, Millipore), phospho-Ser473-AKT (1:2000), phospho-Thr308-AKT (1:1000), AKT (1:1000), phospho-ERK 1/2 (1:2000), ERK 1/2 (1:1000) (all these antibodies from: Cell Signaling Technology), and β-Actin (1:10000, Sigma). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:10000, Calbiochem). Antigen-antibody complexes were visualized using Immobilon Forte Western HRP Substrate (Millipore) and ImageQuant LAS 4000 (GE Healthcare). Band intensity was quantified using ImageJ software.

Cells were suspended in lysis buffer (20 mM tris pH 7.5, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 1% NP-40, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin) and 20 μg of each fraction was separated by SDS-PAGE, followed by western blot analysis. The membranes were incubated with antibodies; phospho-Ser⁴⁷³Akt, phospho-Thr³⁰⁸Akt, phospho-Ser^632/635IRS-1, phospho-ERM, and ERM from Cell Signaling Technology (Beverly, Massachusetts); Akt, ROCK1 (H-85), ROCK2 (H-85), PPARγ, α-actin, phosphor-cofilin, GAPDH and monoclonal antibodies specific for RhoA (26C4) from Santa Cruz Biotechnology (Dallas, Texas); phospho-Y⁶¹² IRS-1 (Invitrogen); phospho-Thr⁸⁵³ MYPT1 was from CyclexMBL (Japan). The bands were visualized with a ChemiDoc imaging system (Bio-Rad) and quantified by Image Lab software (Bio-Rad) and ImageJ software (National Institutes of Health, ver.1.51k).

Phospho-Thr172-AMPK, AMPK, phospho-Ser79-ACC, ACC, PPARγ, C/EBPα, phospho-Thr308-Akt, and Akt were purchased from Cell Signaling Technology. Srebp-1 and Actin were purchased from Santa Cruz Biotechnology, and horseradish peroxide (HRP)-conjugated donkey anti-rabbit IgG was obtained from GE Healthcare.