Lck biosensors with different substrates were constructed using a published Src biosensor as the template (31), replacing the Src-specific substrate with Lck-specific substrates. For bacterial expression, the Lck biosensors were inserted into a pRSET-B backbone plasmid and transformed into BL21 competent E. coli cells for protein expression. For the mammalian cell expression, Lck biosensors were inserted into a customized pCAGGS vector using Gibson Assembly (New England BioLabs) (46). The biosensors were transformed into DH5α competent E. coli cells for DNA amplification. LckWT and Lck mutants were inserted into a customized pCAGGS vector. The initial template constructs LckWT-CFP, LckK273R-CFP, and LckY394F-CFP were gifts from Dr. Katharina Gaus (Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia). For lentiviral production, fragments of the ZapLck biosensor, LckWT, LckY394F, and LckK273R, were amplified using polymerase chain reaction (PCR) and then inserted into a pSIN vector between Spe I and Eco RI restriction enzyme sites. For expression level experiments, LckWT and LckY394F were fused with mCherry at the C terminus through overlap PCR and inserted into the pSIN vector for lentiviral production. For visualization and analysis of Lck-Lck interaction, LckWT and LckY394F were fused with VN173 and VC155, respectively, at the C termini and inserted into the pSIN vector through Gibson Assembly. All the fragments were amplified using Q5 high-fidelity DNA polymerase (New England Biolabs, cat#M0491L). The pBiFC-bJunVN173 (cat#22012) and pBiFC-bFosVC155 (cat#22013) used as templates of VN173 and VC155 were purchased from Addgene.

To amplify the viruses, human embryonic kidney (HEK) 293T cells were cotransfected with the target plasmids contained in the pSIN vector, pCMVΔR, and pCMV-VSVG using Lipofectamine 2000 (Invitrogen) in Dulbecco’s minimum essential medium (DMEM) containing 10% fetal bovine serum (FBS). Eight hours later, we changed the medium with advanced DMEM containing 2% FBS and 0.2% penicillin-streptomycin. After 48 hours, we collected the virus and purified the sample with a 0.22-μm filter. To infect Jurkat or JCam cells, the virus was added to cells in a 1:1 volume ratio (with cell concentration below 1 × 106 cells/ml). For JCam cells infected with LckWT, LckY394F, or LckK273R, we used puromycin (1 μg/ml) to select positive cells over a duration of 1 week before imaging.

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