Fig. 6: Identification of a potential binding interface between SSL10 and TNFR1.
a Immunoblotting of SSL10 after pull-down with MBP-TNFR1ECD or its four deletion mutants (i.e., ΔCRD1-4). The relative band intensities of SSL10 proteins pulled down by WT or mutant MBP-TNFR1ECD are quantitated by densitometry after normalization to their input, and then expressed as the fold of WT SSL10 pulled down by WT MBP-TNFR1ECD. b ITC assays of SSL10 binding to the TNFR1ECD mutant ΔCRD1 or ΔCRD2. c LDH released from HUVEC cells treated with 2 μM SSL10 alone or combined with 10 μM MBP-TNFR1ECD, ΔCRD1 or ΔCRD2, as indicated. d Model 1 of SSL10/TNFR1ECD complex was generated by HawkDock program with the crystal structures of SSL10 and TNFR1ECD (PDB code: 1EXT). The enlarged view shows the residues of SSL10 predicted to interact with TNFR1ECD. SSL10 and TNFR1ECD are colored cyan and light brown, respectively. e The electrostatic surface view of predicted binding site of SSL10 for TNFR1ECD is shown. The positive and negative charge are colored blue and red, respectively. f Immunoblotting of SSL10 and its mutants (M1 and M2) after pull-down with MBP-TNFR1ECD. g ITC assay of mutant M1 binding to MBP-TNFR1ECD. h LDH released from HEK293T cells treated with 2 μM SSL10 or mutant M1. All data represent the means ± SD calculated from three independent experiments. *p < 0.05; **p < 0.01, ***p < 0.001 compared to the Ctrl cells (buffer-treated cells) or as indicated. n.s. not significant, by one-way ANOVA.
