Fig. 4: TNFα cleavage altered by N-terminal SPPL chimeras.

a DKO cells (ctrl.+) and dKO cells overexpressing the indicated wt or chimeric proteases were transiently transfected with full length TNFα (TNFα FL). Non-TNFα transfected cells. (ctrl -) were included. Membrane bound TNFα species were analyzed on Western Blot using the anti-FlagM2 antibody. An antibody against the HA-tag (3F10) was used to detect SPPL expression. b, c Densitometric quantification of non-cleaved TNFα NTF, as depicted in (a). Normalization to TNFα FL eliminated transfection variations. Expression differences between chimeric and wt proteases are eliminated by multiplication with the expression factor (ExF). Resulting values were all normalized to their corresponding wt protease sample. (d&e) Densitometric quantification of total TNFα ICD, as depicted in (a). Normalization as in (b, c). Expression differences between chimeric and wt proteases are eliminated by division with the ExF. b–e 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 2b/2a = 0.1191, p of 2c/2a = 0.0041, p of 3/2a = 0.0002; c: p of 2a/2b = 0.0031; d: p of 2b/2a = 0.8154, p of 2c/2a = 0.0011, p of 3/2a = 0.8419; e: p of 2a/2b = 0.8872), n = 6 independent experiments. The ExF is the mean (n = 6) of the ratio between the expression of the individual chimera and its corresponding wt protease.