Ab91109

Different CD4⁺ T cells, as indicated above, were lysed using RIPA lysis buffer with a protease inhibitor to obtain total proteins (Beyotime, China). The protein was separated in SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Thereafter, the primary antibody was applied at 4°C overnight. The secondary antibody (Beyotime, Shanghai, China) was applied for 1 h at 37°C. The protein expression was detected after the application of ECL substrate. All the data represent the average of three replicates. Our study used the following primary antibodies: TSG101 (Ab125011, Abcam), CD63 (Ab59479, Abcam), CD81 (Ab109201, Abcam), SERPINB2 (Ab269275, Abcam), T-bet (Ab91109, Abcam), GATA-3 (Ab106625, Abcam), and β-actin (20536-1-AP, Proteintech).

The process of immunofluorescence analysis was performed as described by our previous studies (Yin et al. 2013a (link)). Briefly, brain slices were permeabilized with 0.3% Triton-X 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. The brain slices were not treated with Triton-x 100 when staining with anti-GAD67 antibodies. After washing with PBS for three times, samples were incubated with Alexa Fluor-488 or -405 secondary antibodies (1:1000, Invitrogen) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-Drd2 (1:200, Millipore, AB5084P), rabbit anti-NeuN (1:500, Abcam, ab177487), mouse anti-PV (1:500, Sigma, P3088), mouse anti-GAD67 (1:300, Millipore, MAB5406), mouse anti-TBX21 (1:100, Abcam, ab91109), and rabbit anti-OMP (1:200, Abcam, ab183947).

Samples were lysed with RIPA buffer and centrifuged (10 min, 1,049x g) to remove cell debris. Protein concentrations were determined by BCA assay (Beyotime Biotechnology, China). Equal amount of proteins (20 µg) was subsequently separated by 12% SDS-PAGE and transferred to PVDF membrane, which was blocked with 5% skimmed milk and incubated with primary antibodies overnight. After washing with PBS with Tween 20 (1 hr, room temperature) and incubation overnight at 4°C with the appropriate primary antibody, membranes were incubated with secondary antibodies for 2 hr. The following antibodies were used: T-bet (1 : 1,000, ab91109, Abcam), GATA-3 (1 : 1,000, ab214804, Abcam), IL-2 (1 : 200, ab231441, Abcam), IL-4 (1 : 1,000, ab11524, Abcam), IFN-γ (1 : 4,000, AMC4739, Invitrogen), IL-10 (1 : 1,000, ab189392, Abcam), second antibody (1 : 2,000, CST 7074), and β-actin (1 : 2,000, TA-09, Zhongshan Company). Protein bands were visualized using a ChemiDoc Touch imager (Bio-Rad, USA). The gray area ratios of different bands were calculated by densitometry using ImageJ v1.8.0 software (National Institutes of Health). Each experiment was performed in duplicate.