For the stimulation, different regulators and their combinations were employed. An antibody targeting CD3, the upstream ZAP-70 recruiter in the TCR signalling cascade can be used to stimulate TCR signalling with increased phosphorylation activities. Hydrogen peroxide is a reactive oxygen species, activating the response of extracellular receptor kinases via oxidative stress, which in turn initiates TCR signaling and activation of ZAP-70. Finally, sodium pervanadate is an inhibitor of phosphotyrosyl protein phosphatases (PAP) and hence, it can be used to generally boost protein phosphorylation. The cells were stimulated using individual regulators or combinations of regulators as shown in Fig. thereafter lysed and the lysate subsequently analysed by PAGE and WB using the anti-phospho ZAP 70 antibodies.