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. Author manuscript; available in PMC: 2023 Sep 1.
Published in final edited form as: Nat Chem Biol. 2023 Feb 13;19(3):378–388. doi: 10.1038/s41589-022-01253-7

Extended Data Fig. 7. Characterization of C20:4 PC-dependent acyltransferase activity of TMEM164.

Extended Data Fig. 7

a, Western blot of membrane lysates from sgCtrl and sgTMEM164-2 786-O cells recombinantly expressing C-terminally FLAG-tagged WT-TMEM164 or a C123A-TMEM164 mutant. WT-TMEM164 lysates were serially diluted with mock lysate to identify a dilution (1:1) that matched the expression of C123A-TMEM164. Data are from a single experiment representative of two independent experiments. b, Membrane lysates from sgCtrl or sgTMEM164 786-O cells were treated with N-ethylmaleimide (NEM; 200 µM, 60 min) prior to exposure to diacyl PC(C18:0/C20:4-d8) and lyso-ePE-P(C18:0) substrates and measurement of ePE-P(C18:0/C20:4-d8) product. c, d, Measurement of (c) ePE-P(C18:0/C20:4-d8) and (d) diacyl PE(C17:1/C20:4-d8) formation in membrane lysates from sgCtrl and sgLPCAT3 786-O cells using diacyl PC(C18:0/C20:4-d8) and ePE-P(C18:0) or LPE(C17:1) as donor and acceptor substrate, respectively. For b-d, membrane lysates (1 µg/µL) were treated with 50 µM each of donor and acceptor substrates for 1 h at 37° C prior to analysis. For b-d data represent mean values ± S.E.M from three independent experiments per group. P-values were derived using a Two-sided Student’s t-test performed relative to sgCtrl cells.