α ha magnetic beads

To detect the interactome of RagA protein in physiological condition, naïve CD4⁺ T cells from C57BL/6 or HA-RagA knock-in mice were cultured in plates coated with α-CD3-CD28 mAb (10 μg/ml each) for 4.5 days in Click’s medium containing IL-2 and TGF-β for differentiation into Treg cells. For immunoprecipitation, α-HA magnetic beads (Thermo Fisher Scientific) was used to pull down HA-RagA. Treg cells were lysed in IP lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA) and applied to beads, incubated at 4 ^°C under rotation for 3 h and washed at least 3 times. Lammaeli buffer (2×) were added to each tube containing the beads and boiled for 5 min to release the bound proteins. Immunoprecipitated proteins were digested and the peptides were labeled with individual TMT reagents and pooled, followed by basic pH reversed phase LC fractionation. Each fraction was then analyzed using acidic pH reverse phase nanoscale LC-High Resolution MS/MS. Protein detection and quantification and computational analysis were performed as we described previously (Shi et al., 2018 (link)).

For immunoprecipitation, 1 × 10⁸ transduced cells were lysed in 100 μl CHAPS lysis buffer (0.3% CHAPS, 40 mM HEPES pH 7.4, 120 mM NaCl, 20 mM NaF, 1 mM EDTA). About 2–4 μg of the indicated antibodies were bound to 5 μl of protein A/G agarose (sc-2003; Santa Cruz) and then added to the cleared cellular lysates and incubated with rotation for 2 h. Immunoprecipitates captured with protein A/G agarose were washed three times with the CHAPS lysis buffer and eluted with 2× sample reducing buffer (39000; ThermoFisher), followed by immunoblot analysis. The following antibodies or antibody-conjugated beads were used for immunoprecipitation: α-Sec31a (17913-1-AP; ProteinTech), α-Sec13 (GTX101055; GeneTex), α-Skp1 (12248; Cell Signaling Technology), α-Wdr24 (20778-1-AP; ProteinTech), and α-HA magnetic beads (88837; ThermoFisher).