Fig. 5: NEDD4L promoted ubiquitination of SLC39A14, SLC39A8, and STEAP3.

A–C Ubiquitination of endogenous SLC39A14 (A), SLC39A8 (B), and STEAP3 (C) upon NEDD4L overexpression in NK-92 cells (n = 3). Ubiquitination of endogenous SLC39A14 (D), SLC39A8 (E), and STEAP3 (F) upon NEDD4L knockout in NK-92 cells (n = 3). NK-92 cells were co-transfected with His-tagged SLC39A14 (G), SLC39A8 (H), or STEAP3 (I), HA-tagged ubiquitin, and Myc-tagged WT or HECT deletion mutant of NEDD4L, and the ubiquitination of substrates was analyzed (n = 3). NK-92 cells were co-transfected with His-tagged SLC39A14 (J), SLC39A8 (K), or STEAP3 (L), HA-tagged ubiquitin, and Myc-tagged NEDD4L WT or C2 deletion mutant, and the ubiquitination of substrates was analyzed (n = 3). Effects of Heclin on NEDD4L-mediated ubiquitination of SLC39A14 (M), SLC39A8 (N), and STEAP3 (O) in NK-92 cells (n = 3). Ubiquitination of five His-tagged lysine mutants of SLC39A14 (P), one His-tagged lysine mutant of SLC39A8 (Q), or six His-tagged lysine mutants of STEAP3 (R) in NK-92 cells (n = 3). S Sequence alignment of SLC39A14, SLC39A8, and STEAP3 from different species showing the conservation of K267, K284, and K484 sites, respectively. T Immunoblotting analyzing the expression of three proteins in NK-92 cells transfected with His-tagged WT or lysine to arginine mutants of SLC39A14, SLC39A8, and STEAP3, and treated with CHX (n = 3). Determination of ubiquitinated chain linkages of SLC39A14 (U), SLC39A8 (V), and STEAP3 (W) in NK-92 cells. X Immunoblotting showing the effect of NEDD4L on K48 or K63 ubiquitin linked to SLC39A14 or SLC39A8, and K63 ubiquitin linked to STEAP3 in NK-92 cells (n = 3). T represented mean ± SD analyzed by unpaired t test. *P < 0.05, **P < 0.01. CHX Cycloheximide, WT wild-type.