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. 2024 Jun 17;300(7):107480. doi: 10.1016/j.jbc.2024.107480

Figure 8.

Figure 8

hPLA2R1 does not appear as a substrate for β-secretase and γ-secretase in HEK293 cells and MEFs.A–C, hPLA2R1 expression was induced in T-Rex-293 cells by tetracycline treatment (1 μg/ml) for 48 h. A, medium was changed and cells were pretreated for 30 min with the β-secretase BACE-1 inhibitor (Bi, 100 μM), the ADAM10 inhibitor GI254023X (GI, 10 μM) or both, then vehicle or PMA (200 nM) was added for 1, 2, or 3 additional hours. Upper panels, WB of medium from unstimulated and PMA-stimulated cells probed with anti-PLA2R1 antibodies targeting the extracellular region of hPLA2R1 to detect Shed-PLA2R1. Lower panel, WB of cell lysate with anti-HA antibodies to detect FL-PLA2R1 and Cter-PLA2R1. B, medium was changed and cells were pretreated with GI (10 μM) and/or the indicated concentrations of Bi for 30 min, then vehicle or PMA (200 nM) was added for 1 h. Immunoblots of medium (upper panel) and cell lysate (lower panels) were probed with anti-PLA2R1 antibodies targeting the extracellular region of hPLA2R1 to detect Shed-PLA2R1 or anti-HA antibodies to detect FL-PLA2R1 and Cter-PLA2R1. Actin was used as a loading control. C, medium was changed and cells were pretreated with GI (30 μM) or the γ-secretase inhibitor DAPT (10 μM) for 1 h. Immunoblots of medium (upper panel) and cell lysate (lower panels) were probed with anti-PLA2R1 antibodies targeting the extracellular region of hPLA2R1 to detect Shed-PLA2R1 or anti-HA antibodies to detect FL-PLA2R1 and Cter-PLA2R1. Actin was used as a loading control. D, HEK293 cells were transfected with Δ7 and treated for 1 h with vehicle (Ctrl), GI (30 μM), DAPT (10 μM), or Bi (30 μM) (upper panel) and with vehicle or D6 inhibitor (12.5 μM) (lower panel). Protein samples from cell lysate were analyzed by WB with anti-HA antibodies to detect the full-length form of Δ7 and Cter-PLA2R1. E, mouse embryonic fibroblasts (MEFs), either WT or KO for presenilins (PS1/PS2 double KO) were transfected with Δ7 for 48 h. Protein samples from cell lysate were analyzed by WB with anti-HA antibodies to detect full-length Δ7 and Cter-PLA2R1, respectively. WT MEFs were also transfected with Δ7 and treated with the above inhibitors. F, schematic representation showing that hPLA2R1 is not cleaved by β-secretase and γ-secretase in HEK293 cells and MEFs in our settings.