LOX-1 NTFs undergo SPPL2a/b-dependent intramembrane proteolysis. (A) HeLa cells were transfected with HA-LOX-1 and WT or inactive (D/A) SPPL2 proteases. Where indicated, SPPL2a/b activity was inhibited with 20 µM ZLL for 6 h. (B) Quantification of A. N = 7, n = 7. Student’s t test. Norm., normalized. (C) Colocalization of HA-LOX-1 and SPPL2a-myc or SPPL2b-myc in transfected HeLa cells. (D and E) iMAECs transfected with HA-LOX-1 were treated with 40 µM ZLL for 24 h before Western blot analysis. LOX-1 (NTF1+2)/FL ratios are depicted in E. N = 2, n = 5. Student’s t test. (F and G) WT or SPPL2a/b dKO MEFs were stably transduced with HA-LOX-1-FLAG followed by Western blot analysis. LOX-1 (NTF1+2)/FL ratios are depicted in G. N = 2, n = 6. Student’s t test. (H) WT, SPPL2a- SPPL2b-, or double-deficient MEFs were transfected with HA-LOX-1 and analyzed by Western blotting. (I) Quantification of H. N = 3–4, n = 5–8. One-way ANOVA with Tukey’s post hoc testing. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant. N, the number of independent experiments; n, the number of individual samples for quantification. All data are shown as mean ± SD.