Pi3k p110α

Cells and LUAD samples were harvested in RIPA buffer (Beyotime, Shanghai, China) and centrifuged for 10 min at 12,000 × g and 4 °C. Then supernatants were collected, and protein concentrations were calculated by the BCA Kit (Ysasen, Shanghai, #B68010). We took 40–60 μg per sample, and performed protein separation by SDS-PAGE electrophoresis and transferred gel onto PVDF membrane. After blocked by the 5% milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies against Mex3a (Abcam, #ab79046), GAPDH (Cell Signaling Technology, #5174), LAMA2 (Abcam, #ab236762), E-cadherin (Cell Signaling Technology, #14472), N-cadherin(Cell Signaling Technology, #13116), Snail (Cell Signaling Technology, #3879), Slug(Cell Signaling Technology, #9585), PI3K p110α (Cell Signaling Technology, #4249), pAKT (Cell Signaling Technology, #4060), and AKT (Cell Signaling Technology, #4691) at 4 °C overnight. The membrane was incubated with a secondary antibody at room temperature for 1 h After three washes with TBST. Then, the signals were detected by enhanced chemiluminescence following the manufacturer’s recommendations.

Total protein was extracted in radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing 1 × Halt Protease and phosphatase inhibitors (Thermo Scientific, USA). For western blotting, equal amounts of total proteins were electrophoresed by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The antibody for NRAS (ab77392) was purchased from Abcam (USA), and antibodies for AKT (#9272), pAKT-ser473 (#4060), PI3 K-p110α (#4249) and PI3 K-p110β (#3011) were purchased from Cell Signaling (USA). The members were probed with primary antibodies overnight at 4°C. Following three washes with tris-buffered saline with Tween (TBS/T) buffer, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulin G (IgG) for 1 h at room temperature. EZ-ECL (Beit-Haemek, Israel) was subsequently used for visualization of the bands. The membranes were stripped and probed with the GAPDH monoclonal antibody (KangChen, China) as the control. Changes in protein levels were measured relative to the internal control GAPDH level, and fold changes were obtained relative to values for the respective negative control (NC). All experiments were repeated at least three times.

Fisetin (purity ≥ 98%), thiazolyl blue tetrazolium bromide (MTT) and mouse monoclonal β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib Tosylat N Mikron (BAY 43-9006) was provided by Bayer HealthCare (Bayer Pharma AG, Berlin, Germany). Dead cell apoptosis kit with annexin V alexa fluor^® 488 and propidium iodide (PI) were obtained from Life Technologies (Grand Island, NY, USA). Rabbit monoclonal or polyclonal antibodies for PARP, cleaved caspase-3, Bcl2, Bax, Bak, Mcl-1, PI3Kp110α, PI3Kp85, pAKT Ser⁴⁷³, pmTOR Ser²⁴⁴⁸, pmTOR Ser²⁴⁸¹, pMEK1/2 and pERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal cyclin D1 antibody was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Rabbit polyclonal Ki67, goat polyclonal PNCA, goat polyclonal PECAM-1/CD31 and mouse monoclonal VEGF antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase conjugates goat anti-rabbit, rabbit anti-goat and rabbit anti-mouse antibodies were purchased from Millipore Corporation (Billerica, MA, USA). Alexa Fluor 488 rabbit anti-goat antibodies were procured from Life Technologies (Grand Island, NY, USA).

Ex vivo post-cataract surgery cultures were extracted on Time 0. For western blot analysis of PI3K p110 isoform expression, 10–20 μg of protein was loaded per lane and separated by 4–12% SDS-PAGE and transferred at 4° on PVDF membranes. Membranes were blocked using 5% milk for 1 h and incubated overnight at 4° with primary antibodies: PI3K p110α (4249, Cell Signaling, Danvers, MA, USA), p110β (sc-376641, Santa Cruz, Dallas, TX, USA), and p110γ (sc-7177, Santa Cruz, Dallas, TX, USA). Following primary antibody incubation, HRP-conjugated mouse, or rabbit secondary antibodies (Bio-Rad, Hercules, CA, USA) were incubated for 1hr, and membranes were exposed to ECL+ chemiluminescence-substrate-detection reagent using ProteinSimple machine. For Wes protein analysis ex vivo post-cataract surgery cultures were extracted on day 3 and 1 μg of protein was loaded per well. For analysis of pAKT and total AKT, phosphor-AKT (4060, Ser473, Cell Signaling, Danvers, MA, USA) and AKT antibody (9272, Cell Signaling, Danvers, MA, USA) were added and the protocol was followed per manufacturer’s instructions using Simple Western Automated Western Blot Wes Instrument. (Biotechne, Minneapolis, MN, USA) as previously described [31 (link)].

3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33258 and cisplatin were obtained from Sigma (St. Louis, MO, USA). The annexin V-FITC apoptosis detection kit and cell cycle analysis kit were purchased from Beyotime (Invitrogen, Carlsbad, CA, USA). Precision Plus Protein Standards (Dual Color) and Immun-Star™ WesternC™ Chemiluminescent Kit were purchased from Bio-Rad (Hercules, CA, USA). Antibodies against procaspase 8, cleaved caspase 9, cleaved PARP, PI3K p110α, Akt, p-Akt (Thr³⁰⁸ and Ser⁴⁷³), mTOR and p-mTOR (Ser²⁴⁴⁸ and Ser²⁴⁸¹) were obtained from Cell Signaling Technology (Beverly, MA, USA). All other primary antibodies, as well as anti-rabbit and anti-mouse secondary horseradish peroxidase antibodies, were purchased from Abcam (Cambridge, MA, USA). All other common chemicals were reagent grade.

Total proteins were extracted from the cells using T-PER protein extraction reagent (Pierce Chemical Co., Rockford, IL). The protein was subjected to 10% SDS-polyacrylamide electrophoresis, and then electrophoretically transferred on to a nitrocellulose membrane. The membrane was incubated with primary antibodies against Akt (1∶1000), p-Akt (Ser473) (1∶1000), S6 (1∶500), p-S6 (1∶500), mTOR (1∶500), p-mTOR (Ser2448) (1∶500; Cell Signaling Technology), p-mTOR (Ser2481) (1∶500), p-ERK1/2 (1∶500), PI3K p110α (1∶500), PI3K p85 (1∶500), 4E-BP1 (1∶1000), p-4E-BP1 (T37/46) (1∶1000), cleaved caspase 3 (1∶400), LC3 (1∶500; Cell Signaling Technology), and Rictor (1∶500). The protein expression was detected using an EnVision+system (DakoCytomation, Glostrup, Denmark). 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen. Semiquantitative analysis of the results was performed for three independent experiments using NIH J image software (National Institutes of Health, Bethesda, MD).