Fig. 2.

Immunoblot validation of novel SPPL3 candidate substrates identified in HEK293 cells overexpressing catalytically active SPPL3. Levels of secreted (s) and cellular XylT2 (A), HS6ST2 (B), HS6ST1 (C), β3GalT6 (D), Sgk196 (E), and EXTL3 (F) were analyzed by immunoblotting in TCA-precipitated conditioned supernatants (sup) and whole-cell lysates, respectively, obtained from HEK293 cells. Where indicated, cells were transiently transfected with nontargeting control siRNA pools (CTRL) or with siRNA pools specific for SPPL3 (20 nm). siRNA pools targeting the individual substrates were transfected to control for antibody specificity. SPPL3 was ectopically expressed in a HEK293 cell line stably transfected with SPPL3 under control of a doxycycline-sensitive repressor. To induce SPPL3 overexpression, media were supplemented with doxycycline (+ Dox) throughout the experiment, whereas control cells were left untreated (− Dox). In all panels calnexin was used as a loading control. **, Sgk196 specific band of unknown nature.