Anti stii antibody

Protein G Dynabeads (ThermoFisher) were incubated with anti-STII antibody (GenScript) for 60 minutes, cross-linked for 30 minutes using 20mM dimethyl pimelimidate (ThermoFisher) diluted in 200 mM triethanolamine (Fisher Scientific), quenched with 150mM monoethanolamine (Fisher Scientific), and washed three times with 1× PBS. T cells were lysed in NP40 Cell Lysis Buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors. Lysates were incubated on ice for 15 minutes and then centrifuged at 10,000×g and 4°C for 10 minutes. Immunoprecipitations were performed according to manufacturer’s instructions where Dynabeads were incubated with equal masses of cleared lysates for 90 minutes at room temperature.

Protein G Dynabeads (ThermoFisher) were incubated with anti-STII antibody (GenScript) for 60 minutes, cross-linked for 30 minutes using 20mM dimethyl pimelimidate (ThermoFisher) diluted in 200 mM triethanolamine (Fisher Scientific), quenched with 150mM monoethanolamine (Fisher Scientific), and washed three times with 1× PBS. CAR T cells that had previously been stimulated with ROR1 beads for 45 minutes were lysed as previously described. Immunoprecipitations were performed according to manufacturer’s instructions where Dynabeads were incubated with equal masses of cleared lysates for 90 minutes at room temperature.