Human FcγRIIA or FcγRIIA with mutations in the ITAM motif (Tyr299 and 304) and the cytoplasmic tail (Tyr281)(ΔITAM-FcγRIIA) were generated by site directed mutagenesis and cloned into the lentiviral plasmid pWPI (modified from the Addgene, plasmid #12254 by removing the EGFP cassette). HEK293T cells (ATCC) were transfected with the lentiviral construct (pWPI) and plasmids psPAX2 and pMD2.G (Addgene, #12260 and #12259) using Lipofectamine (Invitrogen). Supernatant of transfected cells were used to transduce HL60 cells (ATCC) or Jurkat cells as described (Saggu et al., 2018 (link)). Cell lines were sorted on a BD FACS Aria to obtain populations with similar levels of FcγRIIA.
Sequence-verified small hairpin RNA (shRNA) lentiviral plasmids targeting Lyn (Sigma-Aldrich, TRCN0000230901, and TRCN0000218210) or SHP-1 (Sigma-Aldrich, TRCN0000235432, and TRCN0000244305) were used. Lentiviral plasmids SHC001 and SHC002 (Sigma-Aldrich) were used for generating Lyn- and SHP-1-shRNA control lines, respectively. HEK293T cells were transfected with lentiviral construct as described above. Viral transduced HL60–2A cells were then cultured for 48 hours and selected with 6.0 μg/mL puromycin for 5 days in RPMI 1640 with 10% FBS, L-glutamine, and penicillin/streptomycin. Cells were differentiated using 0.8% dimethylformamide (DMF; Sigma-Aldrich) for 4 days.
Sequence-verified small hairpin RNA (shRNA) lentiviral plasmids targeting Lyn (Sigma-Aldrich, TRCN0000230901, and TRCN0000218210) or SHP-1 (Sigma-Aldrich, TRCN0000235432, and TRCN0000244305) were used. Lentiviral plasmids SHC001 and SHC002 (Sigma-Aldrich) were used for generating Lyn- and SHP-1-shRNA control lines, respectively. HEK293T cells were transfected with lentiviral construct as described above. Viral transduced HL60–2A cells were then cultured for 48 hours and selected with 6.0 μg/mL puromycin for 5 days in RPMI 1640 with 10% FBS, L-glutamine, and penicillin/streptomycin. Cells were differentiated using 0.8% dimethylformamide (DMF; Sigma-Aldrich) for 4 days.