All flow cytometric data were collected on a FACS Calibur or FACS LSR II system (both from BD Biosciences) and analyzed using FlowJo analysis software v7.6.1. For intracellular cytokine staining, cells were stimulated for 5 hours at 37°C with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ionomycin (1 mg/ml; Sigma-Aldrich), and brefeldin A (eBioscience). After staining for surface markers, cells were fixed and permeabilized according to the manufacturer’s instructions (eBioscience). Intracellular staining was processed using intracellular fixation buffer (eBioscience), and a TF staining buffer set (eBioscience) was used for RORγt and Foxp3 staining. For the detection of phosphorylated STAT3 by flow cytometry, BD Phosflow Fix Buffer I and Perm/Wash Buffer I were used.

The following antibodies (clone names are in parentheses) with different fluorochrome labels were purchased from eBioscience: CD4 (RM4-5), CD8a (53-6.7), TCRβ (H57-597), CD44 (IM7), CD62L (MEL-14), IFN-γ (XMG1.2), IL-17A (TC11-18H10.1), IL-4 (11B11), and RORγt (B2D). The following reagents were purchased from BioLegend: IL-23R (12B2B64), IL-21R (4A9), CD126 (D7715A7), Foxp3 (MF-14), and IL-17F (9D3.1C8).

For Western blot and ChIP, anti-Cxxc1 (1:1000 dilution for Western blot; 6 μg for each immunoprecipitation and ChIP reaction; ab56035) was purchased from Abcam. H3K4me3 (4 μg for each ChIP reaction; 39915) was purchased from Active Motif. Anti–pC-SMAD2 (3101), anti-SMAD2 (3103), anti-SMAD3 (9523), anti–pC-SMAD3 (9520), anti-STAT3 (Tyr705) (9131), anti-STAT3 (9132), anti-Erk (Thr202/Tyr204) (4370), anti-Erk (4695), anti-JNK (T183/Y185) (9251), and anti-JNK (9258) were obtained from Cell Signaling Technology.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.