Rat anti il 17a

The cells were treated as described in the “Flow cytometry” section. Six hours before the end of the treatment, the cells were stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) plus 1 μg/ml of ionomycin (Alexis, Lausen, Switzerland) for the below experiments. Meanwhile, 10 μg/ml of GolgiStop (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the cells. The treated cells were lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (BioColors, Shanghai, China) containing phenylmethanesulfonyl fluoride. The proteins were analyzed on SDS-PAGE gels and electrophoretically transferred onto the nitrocullose membranes. The membranes were incubated with rabbit anti-RORγt (Santa Cruz biotechnology, Santa Cruz, CA, USA), rat anti-IL-17A (Cell Signaling, Danvers, MA, USA) or goat anti-IL-17F (R&D Systems, Minneapolis, MN) primary antibody respectively overnight. The membranes were washed three times with TBST, followed by the incubation with the appropriate HRP-conjugated secondary antibody for 1 hour at the room temperature. The specific bands were identified with an Enhanced Chemiluminescence Detection Kit (Amersham Biosciences, Piscataway, NJ, USA). This test was repeated for at least three times.

The isolated CD4⁺ T cells were seeded in a 24-well plate at the density of 1.5 × 10⁶ cells/well and treated as described in the “Flow cytometry” section. The In situ expressions of RORγt, IL-17A and IL-17F in the treated CD4⁺ T cells were examined via immunofluorescence staining. The treated cells were fixed in cold methanol at 4°C for 10 min. Then, the cells were washed with PBS and blocked with 5% BSA in PBS for 30 min. The blocked cells were incubated with rabbit anti-RORγt (Santa Cruz biotechnology, Santa Cruz, CA, USA), rat anti-IL-17A (Cell Signaling, Danvers, MA, USA) and goat anti-IL-17F (R&D Systems, Minneapolis, MN) antibodies for 3 h at 4°C. Subsequently, the cells were incubated with a donkey anti-goat IgG-PE antibody (Santa Cruz biotechnology, Santa Cruz, CA, USA), a Alexa Fluor 488-conjugated anti-rat IgG antibody, a Alexa Fluor 488-conjugated anti-rabbit IgG antibody or a Alexa Fluor 647-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Inc.) for 1 h at 37°C. In addition, 6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) staining was performed for 5 min at the same temperature. The cells were detected using the Leica DMRA2 fluorescence microscope with FW4000 software (Leica, Germany). Positive cells or negative cells under 10 fields of view selected randomly for each group were counted for average percentages.