Fig. 3

SPPL2b-mediated intramembrane proteolysis of epithin/PRSS14. a The 427 cells were treated with PMA and/or SPPLs inhibitor, 10 μM (Z-LL)₂ketone and proteolytic fragments were analyzed as in Fig. 2b. b HEK293T cells transfected with FLAG-tagged epithin/PRSS14 and increasing amounts of SPPL2b-HA were analyzed by Western blot using the anti-FLAG antibody to detect NTF and EICD. The expression of SPPL2b and full-length epithin/PRSS14 were also confirmed by Western blot using anti-HA and anti-FLAG antibodies, respectively. Asterisk indicates uncharacterized fragments, presumably resulted from nonspecific cleavages, glycosylation, and/or other modifications of overexpressed epithin/PRSS14. c EICD band intensity was quantified and normalized against tubulin blot and is shown as a bar graph (n = 3). The error bar indicates SD. *p < 0.05, **p < 0.01 (unpaired two-tailed Student’s t test). d The mRNA levels of SPPL2b in SPPL2b-knockdown cell lines (SP2bKD-10 and -16) were detected by real time-PCR. The relative values were normalized to GAPDH signals, as shown in the graphs. Error bars indicate SEM. e Proteolytic fragments of epithin/PRSS14 were analyzed in the control and SP2bKD cells, as in Fig. 2b. f The average of normalized EICD band intensities from three independent immunoprecipitation experiments is shown as a bar graph (n = 3). The error bar indicates SD. *p < 0.05 (unpaired two-tailed Student’s t test). Arrowhead and arrow indicate NTF and EICD, respectively. g Localization of the N-terminal and C-terminal parts of epithin/PRSS14 were analyzed, as in Fig. 1a. Note that the nuclear localization of EICD (arrows) was reduced in SP2bKD-10 cells (arrowheads). Scale bars, 20 μm