Pi3k inhibitor ly294002

The following antibodies were used in this study: anti-phospho-eIF4B (Ser422) (SC-293101, Santa Cruz Biotechnology), anti-eIF4B (SC-376062, Santa Cruz Biotechnology), anti-phospho-STAT5 (Tyr694) (9359L, Cell Signaling), anti-STAT5 (9358S, Cell Signaling), and ani-PDK1 (3062S, Cell Signaling). The inhibitors were purchased as follows: Pim kinase inhibitor SMI-4a (526523, Merck), PI3K inhibitor LY294002 (L9908, Sigma), Akt inhibitor Akti-1/2 (124018, Merck), and mTOR inhibitor rapamycin (SC-3504A, Santa Cruz). All other antibodies and reagents were obtained as previously described [4 (link), 5 (link), 48 (link)].

Recombinant human chemokine CCL27 were from Sino Biological Inc, added to cell cultures at a final concentration of 0.01 to 0.1μg/ml. PI3K inhibitor LY294002 (Sigma Chemical Co., St. Louis, MO) was dissolved in DMSO at a stock concentration of 10 mM and added to cell cultures at a final concentration of 30 μM.

Recombinant human TGF‐β protein was obtained from PeproTech (Rocky Hill, NJ, USA). Primary antibodies against GSK‐3β (27C10), GSK‐3β (Ser9), AKT (C67E7), p‐AKT (Ser473) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against SP1(G447) was obtained from Bioworld (Bioworld Technology, Minneapolis, MN, USA). Protein A/G sepharose and primary antibodies against ubiquitin (SC‐166553) and β‐actin (SC‐130300) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phycoerythrin (PE)‐labelled antibody against ULBP1 and ULBP2 were obtained from R&D (San Diego, CA, USA). Horseradish peroxidase (HRP)‐conjugated secondary antibody, Alexa Fluor 488‐conjugated secondary antibody, 4′, 6‐diamidino‐2‐phenylindole (DAPI) and lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG132 was obtained from Sigma‐Aldrich (St Louis, MO, USA). PrimeScript RT reagent Kit and SYBR Premix Ex TaqTM were products of TaKaRa. E.Z.N.A HP Total RNA Kit was purchased from Omega Bio‐Tek (Doraville, GE, USA). Smart pool siRNA against human SP1 (siSP1), GSK‐3β (si‐GSK‐3β) and control (siNC) were purchased from RiboBio (Guangzhou, China). ERK inhibitor PD98059, p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002 and TGF‐β type I receptor inhibitor SB431542 were obtained from Sigma‐Aldrich.

All chemicals were purchased from Sigma (St. Louis, MO) and antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), respectively, unless specified otherwise. Antibody for HBP1 was from Novus Biologicals (Littleton, CO), and α-tubulin was from Abcam (Cambridge, MA). PVDF membranes and ECL detection reagents for Western blotting analysis were purchased from Perkin Elmer Life Sciences, Inc. (Waltham, MA). Dual-light^® system was from Applied Biosystems (Foster City, CA). PI3K inhibitor LY 294002 was purchased from Sigma (St. Louis, MO). Small interfering RNA (siRNA) molecules specific for HBP1 was purchased from Invitrogen (Carlsbad, CA).

Bovine aortic endothelial cells (BAECs) were purchased from Lonza (Walkersville, MD, USA), and cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) (Invitrogen). Human aortic endothelial cells (HAECs) (Lonza), were cultured in endothelial basal medium2 (EBM2) (Lonza) containing 2% FBS with growth supplements. The cells were passed every 4–5 days, and experiments were performed on either third or fourth passage of cells. After cells had grown to confluence, they were placed in a starvation medium (0.5% FBS) for 16 h. Starved cells were pretreated for 1 h with vehicle or selective 25 μM PI3-K inhibitor LY294002 (Sigma, St. Louis, MO, USA), 10 μM MEK inhibitor U0126 (Calbiochem, Billerica, MA, USA), 20 μM selective p38 MAP kinase inhibitor SB203580 (Alexis, San Diego, CA, USA), 20 μM selective JNK inhibitor SP600125 (Alexis) or 20 μM IKK α/β inhibitor Wedelolactone (Calbiochem), followed by treatment with 20 ng/mL TNF-α (Sigma). The concentration of inhibitors was in accordance with doses used in the previous studies [57 (link)–59 (link)].

All the antibodies were from Cell Signaling Technology (Danvers, MA, USA): mTOR (cat #2983), phospho-mTOR (Ser2481, cat #2974), phospho-mTOR (Ser2448, cat #5536), Rictor (cat #9476), phospho-Rictor (Thr1135, cat #3806), Raptor (cat #2280), AKT (cat #4691), phospho-AKT (Ser473, cat #4060), phospho-AKT (Thr308, cat #13038), phospho-AKT (Thr450, cat #9267), GSK-3β (cat #9315), phospho-GSK-3β (Ser9, cat #9336), PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 (Thr37/46, cat #2855), S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K (Thr389, cat #9234), Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen (Carlsbad, CA, USA). Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich (St Louis, MO, USA). Protease and phosphatase inhibitor cocktails were from Calbiochem (San Diego, CA, USA). Cycle Test Plus kit was from BD Bioscience (East Rutherford, NJ, USA). Super Signal West Pico imaging system was from Thermo Scientific (Rockford, IL, USA).