Rabbit anti mouse akt

Treg cells were fixed in 4% paraformaldehyde for 5 minutes, washed with PBS twice, and ruptured in Triton-X-100 at room temperature for 30 min. The cells were incubated for 30 min with immunofluorescent blocking solution. Next, they were incubated at 4°C overnight with different primary antibodies. After washing the primary antibodies, the cells were incubated with different secondary antibodies at room temperature for 1 hour. Then the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and washed with PBS three times; the cells were added to antifluorescence quencher and covered with a cover slip, and the protein expression was detected by Olympus IX73 microscope (Tokyo, Japan). Image J was used for the quantitative analysis. The above antibodies were as follows: primary antibodies: Rabbit anti-mouse β-actin, Rabbit anti-mouse PI3K P110α (all 1 : 100, CST, USA), Rabbit anti-mouse C-JUN antibody, Rabbit anti-mouse AKT, and Rabbit anti-mouse PI3K P85α (all 1 : 100, Abcam, USA); secondary fluorescently labeled antibodies: Donkey anti-rabbit antibody (Alexa Fluor 488) and Donkey anti-rabbit antibody (Cy3) (all 1 : 200, Jackson, USA). Testing equipment was Olympus IX73 microscope (Tokyo, Japan).

Treg cells proteins were extracted according to the Whole Cell Lysis Assay's instructions (KeyGEN BioTECH, Jiangsu, China). We took 50 μg equal amount of protein for electrophoresis and transferred it onto polyvinylidene fluoride membranes. The membranes were blocked at room temperature for 1 h with Nonfat Dry Milk. The membranes were incubated overnight at 4°C with different primary antibodies in Nonfat Dry Milk (CST, USA) configured antibody dilution and incubated at room temperature for 1 h with secondary fluorescently labeled antibodies in the same antibody dilutions. The signal intensity was analyzed by Image J. The above antibodies were as follows: primary antibodies: Rabbit anti-mouse β-actin, Rabbit anti-mouse PI3K P110α (1 : 1000, CST, USA), Rabbit anti-mouse C-JUN antibody, Rabbit anti-mouse AKT, and Rabbit anti-mouse PI3K P85α (1 : 1000, Abcam, USA); secondary fluorescently labeled antibodies: Donkey anti-rabbit antibody (Alexa Fluor 790) and Donkey anti-rabbit (Alexa Fluor 680) (both 1 : 10000, Abcam, USA). The results were tested by Odyssey CLX S/N CLX-0926 (LICOR, USA).