Fig. 9: Non-canonical shedding of TNFα by SPPL3 and chimeric SPPL2 proteases.

a DKO cells (ctrl.) and dKO cells stably overexpressing SPPL3 wt or SPPL2b wt were transiently transfected with full length TNFα (TNFα FL). Membrane bound TNFα species were analyzed as described in Fig. 5. SPPL3 wt was detected using the 7F9 antibody and SPPL2b wt using the 2G8 antibody. Soluble TNFα species were detected from Western Blot of conditioned media (supernatant, Sup.) with the monoclonal V5 antibody. SPPL2b expressing cells serve as a positive control for TNFα ICD production. b Densitometric quantification of non-cleaved TNFα NTF, as depicted in (a). Normalization to TNFα FL eliminated transfection variations. Results are depicted relative to the control cells (dKO). c Densitometric quantification of the SPPL3 non-canonical TNFα shedding product (sTNFα L3), as depicted in (a). Normalization to the ADAM-generated TNFα shedding product (sTNFα) eliminated transfection variations. Results are depicted relative to the control cells (dKO). d The experiment was performed as described in Fig. 4 but includes the soluble TNFα fragments. e, f Densitometric quantification of non-canonical TNFα shedding products (sTNFα L2||L3) generated by either the SPPL2a (e) or SPPL2b (f) chimeric proteases, as depicted in (d). Normalization to the ADAM-generated TNFα shedding product (sTNFα) eliminated transfection variations. Expression differences between chimeric and wt proteases are accounted for by division with the same ExF values as in Fig. 4 (ExF values of 4b, d equals those in (e), ExF values of 4c, e equal those in f). Resulting values were all normalized to their corresponding wt protease sample. Mean + SEM, unpaired, two-tailed one sample t-tests of log-transformed (log₂) values. ns not significant, **p < 0.01, ***p < 0.001 (b: p of 3 wt=0.156; c: p of 3 wt<0.0001; e: p of 2b/2a = 0.9163, p of 2c/2a = 0.269, p of 3/2a = 0.103; f: p of 2a/2b = 0.001), n = 6 independent experiments.