Fig. 2: Tumor LTβR signals by classical NFΚB and nonclassical NFΚB pathways.
From: Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling
a Flow cytometry of LTβR expression on mouse (B16F10) and human (A375) melanoma cells. Median fluorescence intensity (MFI) shown. b, c Immunoblots for classical IKKα/β (p-IKKα/β) (***P = 0.0005) and NFκB-p65 phosphorylation (p-p65) (*P = 0.0109, **P = 0.0021) (b), and nonclassical p100 processing to p52 (*P = 0.0459) (c) in B16F10 pretreated with 20 μM ciLT, nciLT, or control scrambled peptide (CP) for 1 h at 37 oC; and then stimulated with or without agonist anti-LTβR 3C8 mAb (2 μg/mL) for indicated times (b) or 6 h (c). Representative blots shown, quantification of phospho-p65 or p52 and IKKα/β are normalized to p65 and IKKα/β respectively. d Immune precipitation of B16F10 LTβR with anti-LTβR mAb (5G11). Cells stimulated with or without agonist anti-LTβR mAb for 10 min. Representative blots are shown. Quantification of the bound TRAF2 (**P = 0.0004, ***p = 0.0001) or TRAF3 (**P = 0.0004, ***p = 0.0001) is normalized to the loaded LTβR expression. WCL whole cell lysate. e, f Human melanoma A375 cells pretreated with 20 μM human ciLT (hciLT), nciLT, or CP for 1 h at 37 oC; and then stimulated with or without 100 ng/mL human recombinant LTαβ for indicated times (e) or 6 h (f). Immunoblots for p-IKKα/β, p-p65 (nciLT: **P = 0.0019; ***P = 0.0001; ciLT: *P = 0.0407, ***P = 0.0002), and p52 (*P = 0.0242). Each panel is representative of three independent experiments. b–f Mean ± SEM. P values are calculated by two-way ANOVA, Sidak’s multiple comparisons test. ****P < 0.0001. Uncropped gels and Source data are provided as a Source Data file.
