Mouse anti ha antibody

SK-MEL-28 cells stably expressing MAP3K8 were fixed, permeabilized and blocked with 10% goat serum/PBS. Cells were then incubated with primary mouse anti-HA antibody (Cell Signaling #2367) and secondary goat anti-mouse AlexaFuor-94 (Thermofisher) and counterstained with DAPI (0.5 μg/mL) prior to microscopy.

Supernatant was collected and filtered from either 293Glv9 packaging cells or transduced T cells as indicated. To demonstrate E27 binding to PD-1, 293Glv9-PD1 packaging cells were incubated for 24 hours in filtered supernatant from 1928z or 1928z-E27 transduced T cells. To demonstrate that E27 binds to untransduced, bystander cells, 1928z and 1928z-E27 T cells were co-cultured with human T cells transduced to overexpress PD-1, after 4 days stimulation with CD3/CD28 beads, the cells were sorted by flow cytometry following staining with 19E3 mAb to separate CAR⁺ and CAR^- cells. Supernatant or whole cell lysates was loaded onto mini protean TGX gels (BioRad, Hercules, CA, USA) and then transferred to Immun-blot PVDF membranes (BioRad). The was probed with mouse-anti-HA antibody (Cell Signaling,6E2, Cat#2367S) and then goat anti-mouse HRP conjugated antibody (Millipore, GT X MS AP127 Cat#6C0112) for E27. RMP1–14 was detected using a HRP-conjugated mouse-anti-myc tag antibody (Cell Signaling, Cat#2040S). Detection of antibody was achieved with Pierce ECL western blot substrate (Thermo Scientific, Waltham, MA, USA). RMP1–14 scFv co-immunoprecipitation was performed using the Peirce c-Myc Tag IP/Co-IP Kit (Thermo Scientific) according to the manufacturer’s instructions and detected by western blot as described above.