Ecl block

The transduced CAR T cells and CAR Jurkat cells were collected, washed with Dulbecco’s Phosphate Buffered Saline (DPBS, HyClone), and lysed in Laemmli sample buffer supplemented with β-mercaptoethanol (Bio-Rad, Hercules, CA, USA). The samples were boiled for 10 min at 100 °C before loading. The proteins were run on SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Chicago, IL, USA). The membrane was blocked overnight in 1% ECL Block (GE Healthcare). CARs were stained with anti-CD3ζ-chain antibody (Sigma) detected with anti-Rabbit-HRP (GE Healthcare). EYFP was stained with anti-GFP antibody (SCI-Store) detected with anti-Mouse-IgG-HRP (GE Healthcare).

Transduced T-cells and Jurkat cells were washed twice in DPBS (‘HyClone’, Logan, UT, USA), lysed in Laemmli buffer with the addition of β-mercaptoethanol (‘Bio-Rad’, Hercules, CA, USA), and incubated for 10 min at 100 °C. Proteins were resolved using SDS-PAGE techniques and then shifted to a PVDF membrane (‘GE Healthcare’, Chicago, IL, USA). The blocking of non-specific interactions was performed using 1% ECL Block (‘GE Healthcare’, Chicago, IL, USA). Proteins were stained with anti-CD3ζ chain (‘Sigma’, St. Louis, MO, USA), anti-GAPDH (‘Cell Signaling Technology’, Danvers, MA, USA), and anti-EYFP (‘SCI-Store’, Moscow, Russia) antibodies.
Chemiluminescence was detected after incubation of the membrane with anti-rabbit-HRP antibodies (for anti-CD3ζ chain and anti-GAPDH; ‘GE Healthcare’, Chicago, IL, USA) or anti-mouse-IgG-HRP antibodies (for anti-EYFP; ‘GE Healthcare’, Chicago, IL, USA).