Akt p akt

Protein expression was assessed by lysing cells with a high salt lysis buffer containing 500 nM Tris-HCl (pH 7.4), 300 nM NaCl, 1% NP40, 5 mM EDTA, and a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). Equal amounts of total proteins were boiled in sample buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). To detect specific protein expression, the following antibodies (Abs) were used: c-Myc, Survivin, Caspase-3, p27, Tubulin (Santa Cruz Biotech, CA), CXCR4, HMGB1 (Abcam, Cambridge, United Kingdom), AKT, p-AKT, ERK1/2, p-ERK1/2, CD133, Bax, p21 (Cell Signaling Technology, Danvers, MA), Bcl-2 (Dako, Glostrup, Denmark), β-actin (Sigma-Aldrich). Immunoreactive bands were visualized by using HRP-conjugated secondary Ab and the ECL system (Amersham Biosciences, Buckingham, United Kingdom). HMGB1 expression was analyzed in cell supernatants. Briefly, equal amounts of each sample normalized by cell number were separated by SDS-PAGE, and immunoblot analysis was performed with a rabbit polyclonal Ab to HMGB1, as described [18 (link)]. The optical density of the bands was measured by using the National Institutes of Health Image J software (rsb.info.nih.gov/ij).

The degree of phosphorylation of Ser⁴⁷³ on Akt/p-Akt (Cell Signaling Technology, Danvers, MA, USA), Thr¹⁸⁰/Tyr¹⁸² on p38/p-p38 (New England Biolabs, Ipswich, MA, USA), Thr¹⁸³/Tyr¹⁸⁵ on c-Jun N-terminal kinase (JNK)/p-JNK (Abcam) and Thr²⁰²/Tyr²⁰⁴ on extracellular signal-regulated kinase (ERK1/2)/p-ERK1/2 (New England Biolabs) was measured by semi-quantitative western blot analyses in mouse heart and kidney tissue as described previously [20 (link), 25 ] (see ESM Methods for further details).

The western blot was done as previously described [7 (link)]. The primary antibodies for RhoGDI and Akt/p-Akt were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibodies for caspase-3 and GAPDH were acquired from Santa Cruz Biotechnology.

All inhibitors were purchased from Selleck Chemicals (Houston, TX), prepared as 10 mM stock solutions in DMSO, and further diluted with culture medium before use. Annexin V-FITC/PI apoptosis detection kit and cell cycle detection kit were purchased from KeyGen BioTECH (Nanjing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, and Hoechst 33342 were obtained from Solarbio (Beijing, China). Antibodies for Src/p-Src, Mek/p-Mek, Erk/p-Erk, PI3K/p-PI3K, PDK1/p-PDK1, AKT/p-AKT, mTOR/p-mTOR, PTEN/p-PTEN(S380/T382/383), JNK/p-JNK, c-Jun, p38/p-p38, JAK2/P-JAK2, Stat3/p-Stat3, Rb/p-Rb, CDK4, CDK6, Cyclin D1, CDK2, Cyclin E1, RAD51, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-7 were all purchased from Cell Signaling Technology (Danvers, MA, USA). Tubulin and GAPDH primary antibodies were purchased from ZSGB-Biotechnology (Beijing, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse IgG were obtained from BBI life sciences (Shanghai, China).

HMVECs and TALL-104 cells were mixed (1:1) in culture for 12 h. Paraformaldehyde (4%) was added to the culture for 10 min at room temperature prior to cell lysate isolation, as described below.
For immunoblotting, cells were solubilized in ice-cold cell lysis buffer containing 6.52 mg/mL HEPES, 0.42 mg/mL NaF, 8.64 mg/mL NaCl, 0.2 mg/mL MgCl₂, 5% NP-40, and protease inhibitor cocktail (TOPSCIENCE, Shanghai, China). Proteins were fractionated by SDS-PAGE and transferred to PVDF membrane (Millipore, Burlington, MA, USA) and then blocked in 5% milk in PBST. Subsequently, the membrane was incubated overnight at 4 °C with one of a series of primary antibodies against mouse or rabbit PD-L1 (Cell Signaling, Danvers, MA, USA), PD-1 (Cell Signaling), VE-cad (Invitrogen, Waltham, MA, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Syndecan-1 (Santa Cruz Biotechnology), LEF1 (Cell Signaling), YKL-40 (our lab), CCR5 (Abcam, Cambridge, UK), AKT, pAKT (Cell Signaling), ERK, pERK, PI3K (Santa Cruz Biotechnology), Vimentin (Dako), SMa (Abcam), E-cad (Dako, MA, USA), GAPDH (Beyotime), or β-actin (Sigma-Aldrich). The membrane was then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Beyotime). The specific chemiluminescence signal was detected using the ECL reagent (ThermoFisher Scientific).