Engineered ICAM-1 Truncations and Fluorescent Proteins

ICAM‐1‐GFP was described earlier (Kroon et al, 2018 (link)) and cloned into a lentiviral pLV backbone using SnaBI (ThermoFisher, FD0404) and XbaI (ThermoFisher, FD0684)/NheI (ThermoFisher, FD0973). To generate ICAM‐1 truncated sequences, Gibson cloning (NEB) was performed on the pLV‐ICAM‐1‐GFP plasmid. All constructs contain GFP as FP and the ICAM‐1 signal peptide, consisting of amino acids Met1 to Ala27. ICAM‐1 Δ 1 is truncated from Gln28 to Val109; ICAM‐1 Δ 12 has a deletion from Gln28 to Phe212; ICAM‐1 Δ 123 lacks Gln28 to Ile307; ICAM‐1 Δ3 is truncated from Val213 until Ile307; ICAM‐1 Δ 4 lacks Pro311 to Arg391; ICAM‐1 Δ C terminates at Asn504. pLV‐ICAM‐2‐mKate was constructed and packaged by VectorBuilder (Vector ID is VB200624‐1164vtm). pLV‐mNeonGreen‐Caax and pLV‐mScarletI‐CAAX have been described earlier by us (Arts et al, 2021 (link)). mEos4b‐N1 was a gift from Micheal Davidson (Addgene # 54814; http://n2t.net/addgene:54814; RRID:Addgene_54814; Paez‐Segala et al, 2015 (link)). mEos4b was PCRed out of the mEos4b‐N1 vector, after which it was ligated into a pLV backbone using SnaBI and XbaI/NheI. All primers used in cloning are shown in Appendix Table S1.