Macs protein a microbeads

For moesin immunoprecipitation experiments, 5 µl of unconjugated anti-moesin mAb (EP1863Y, Abcam) was mixed with 200 µl of the T cell protein extract (200 µg/ml) and incubated overnight at 4°C; then, 50 µl of µMACS Protein A Microbeads (Miltenyi Biotec) were added (2 h, 4°C). The immunoprecipitate’s washing, elution, and separation were performed according to the manufacturer’s recommendations. For gpALL pull-down, 200 µl of the T cell protein extract (200 µg/ml) was mixed with 30 µL of biotinylated ALL (2.2 mg/ml) and incubated at 4°C overnight; then, 100 µl of Streptavidin MicroBeads (Miltenyi Biotec) were added and further incubated for 2 h, at 4°C; the precipitate’s elution, washing, and separation were performed with 500 mM GalNAc following manufacturer’s instructions. The precipitates were used in later experiments.

Chromatin immunoprecipitation (ChIP) cloning experiments were performed using 1.5×10⁷ CD4 T cells stimulated with IL-2 for 20 min followed by a 10-min crosslink with formaldehyde (0.37%) at 37°C and immediate termination of the reaction by the addition of 1.25 M glycine, as previously described [6] (link). The cells were harvested and shearing of the chromatin was done on a Misonix 3000 sonicator. The sonicated chromatin was immunoprecipitated for an hour using µMACS protein A microbeads (Miltenyi Biotec, UK) and anti-STAT5a and -STAT5b monoclonal antibodies or control mouse IgG Abs (all from Zymed Laboratories, UK). Purified chromatin was blunt-end repaired using T4 DNA polymerase (New England Biolabs, UK), ligated to linkers, PCR amplified, and cloned into TOPO vector (Invitrogen, UK) and sequenced (Cogenics, UK). Validation and confirmation of the GAS sequence within the FRA2 gene, as a STAT5 target was done by quantitative PCR reaction, performed as described in the LowCell# ChIP Kit TM (Diagenode, UK) and data interpreted as a percentage of the starting material (Input).

Cells at a confluence of 70% were lysed with 1 mL ice-cold lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl, pH 8.0) supplemented with protease and phosphatase inhibitors. Following a 30-min incubation period, cells were centrifuged (10,000× g for 10 min at 4 °C) and the supernatant collected. Co-IP experiments were performed as described before [13 (link)]. In brief, to pull down CB, 2–4 µg of rabbit anti-CB (Swant) was used, then 100 µL of µMACS protein A MicroBeads (Miltenyi Biotec, Auburn, AL, USA) were added to the lysate and incubated at 4 °C for 30 min. Samples were loaded on MACS separation columns (Miltenyi Biotec) and subjected to magnetic immunoprecipitation. The columns were washed 3 times with a wash buffer and protein complexes were eluted in 50 µL of pre-warmed SDS gel loading buffer 1X, subjected to electrophoresis (SDS-PAGE) and subsequent Western blotting. Membranes were probed with the antibodies rabbit anti-CB (1:10,000, Swant), rabbit anti-FAK (1:1000, Cell Signaling Technology), and rabbit anti-septin 7 (Bethyl Laboratories Inc., Montgomery, TX, USA); a mouse anti-paxillin antibody (1:2000; BD Bioscience, Allschwil, Switzerland) served as a negative control.