Mtor rabbit mab

MDA-MB-231 cells were seeded on a 6-well plate and treated with F3, lutein, β-sitosterol, or stigmasterol for 8 and 24 h. Cells were harvested and washed with PBS. Cell Fixation and Permeabilization Kits (Abcam, Cambridge, UK) were used for intracellular protein detection. The harvested cells were fixed with a fixation medium (Reagent A) for 15 min, washed with PBS, followed by a permeabilization medium (Reagent B) for 15 min. Next, the cells were washed with PBS and stained with primary antibodies that had been diluted in 3% BSA/PBS for 2 h. The primary antibodies were AKT (pan) Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA), phospho-AKT Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA), mTOR Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA), and HIF1α polyclonal (Thermo Fisher Scientific, Waltham, MA, USA). The incubated cells were then washed with PBS and the Goat Anti-Rabbit IgG H&L (Alexa Fluor) (Abcam, Cambridge, UK) secondary antibody (diluted in 3% BSA/PBS) was added to the cells for 1h. Lastly, the cells were washed with PBS and analyzed using flow cytometry with 10,000 cells per sample detected.

Protein lysates were extracted from brain tissues or culture cells by RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma, USA). Protein concentration was measured by BCA kit (Beyotime, China). A total of 20 μg protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Biorad, USA). Then, the membranes were blocked by 5% fat-free milk for 1 h at room temperature, incubated with first antibodies at 4 °C overnight and corresponding second antibodies for 1 h at room temperature. Then antibodies used in our study were: iNOS Rabbit mAb (CST#13120, 1: 1000), Arginase-1 Rabbit mAb (CST#93668, 1: 1000), GAPDH Rabbit mAb (CST#2118, 1: 1000), LC3A/B Rabbit mAb (CST#12741, 1: 1000), p62 Antibody (CST#5114, 1: 1000), Phospho-Akt (Ser473) Antibody (CST#9271, 1: 1000), Akt Antibody (CST#9272, 1: 1000), Phospho-mTOR (Ser2448) Antibody (CST#2971, 1: 1000), mTOR Rabbit mAb (CST#2983, 1: 1000), pro-IL-1β Rabbit mAb (CST#31202, 1: 1000), Cleaved-IL-1β Rabbit mAb (CST#63124, 1: 1000), pro-Caspase-3 Antibody (CST#9662, 1: 1000), Cleaved Caspase-3 Antibody (CST#9661, 1: 1000), Phospho-GSK-3α/β (Ser21/9) Antibody (CST#9331, 1: 1000), GSK-3α/β Rabbit mAb (CST#5676, 1: 1000) and NLRP3 Rabbit mAb (CST#15101, 1: 1000) were all obtained from Cell Signaling Technology (USA).