Fig. 5: In vitro cleavage of TNFα NTF generated from TNFα FL by SPPL2b chimeric proteases. | Communications Biology

Fig. 5: In vitro cleavage of TNFα NTF generated from TNFα FL by SPPL2b chimeric proteases.

From: The N-terminal PA domains of signal-peptide-peptidase-like 2 (SPPL2) proteases impact on TNFα cleavage

Fig. 5: In vitro cleavage of TNFα NTF generated from TNFα FL by SPPL2b chimeric proteases.The alternative text for this image may have been generated using AI.

(a) DKO cells were either non-transfected (ctrl.) or transiently transfected with SPPL2b wt or the indicated chimeric proteases. In vitro cleavage of separately expressed full length TNFα (TNFα FL) was carried out and TNFα species were immunoprecipitated (FlagM2) and analyzed on Western Blot (polyclonal anti-Flag antibody). SPPL proteases were detected in solubilized membranes with an antibody against the HA-tag (3F10). (b) Densitometric quantification of non-cleaved TNFα NTF, as depicted in (a). Normalization to the antibody signal eliminates experimental variations as described earlier51. Expression differences between chimeric and wt proteases are accounted for by multiplication with ExF. Resulting values were all normalized to the SPPL2b wt protease sample. Mean + SEM, unpaired, two-tailed one sample t-tests of log-transformed (log2) values. ns=not significant, *p < 0.05, ***p < 0.001 (p of 2a/2b = 0.026, p of 2c/2b = 0.0006, p of 3/2b = 0.0242), n = 4 independent experiments. The ExF is the mean (n = 4) of the ratio between the expression of the individual chimera and SPPL2b wt.

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