, UK) were used in Western blot experiments. Antibodies to human CD40 (H-10, Santa Cruz Biotechnology or mAb89 (a gift from Dr. Jacques Banchereau, Baylor Institute for Immunology Research, Dallas)) were used in flow cytometry experiments. Rabbit polyclonal anti-CD40 antibody (Biosource International, USA) was used in immunofluorescence. Supplemental Figure 1: CD154 amplifies XBP-1 mRNA splicing in anoxia-reoxygenation conditions. HK-2 cells were treated for 1 hour with 50 ng/mL antimycin A, a mitochondrial respiratory chain blocking agent, in the presence or not of rsCD154 at a concentration of 100 ng/mL; in these conditions, XBP-1 mRNA splicing was moderately induced but no effect of CD154 on XBP-1 mRNA splicing was detectable (R0). Antimycin A was removed and culture continued in normoxic conditions in the presence or not of rsCD154 and the spliced/unspliced ratio of XBP-1 (XBP1 s/u) mRNA in HK-2 cells measured at the indicated times by RT-qPCR, This work was supported by a grant from MSDAvenir. Supplementary Materials Supplemental Material and Methods: primary antibodies used in this study: antibodies to BiP (Cell Signaling, vol.1, pp.?-tubulin, 842919.
, HK-2 cells were grown under hypoxic conditions for 3 hours in Lumox tissue culture plates (A) or in standard tissue culture plates (B) in the presence or not of rsCD154; IL-6 protein was measured by ELISA in cell culture supernatants (A, n = 5, * significant relatively to control conditions, p.5
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