Fig. 1. APRIL is processed by a furin convertase. (A) Characterization of APRIL transfectants. HEK 293T cells were transfected with APRIL or empty vector (mock); cell extracts were analysed by western blot employing affinity-purified polyclonal anti-APRIL antibody. (B) Immunoprecipitates of culture supernatants derived from APRIL- and mock-transfected cells were analysed by western blot. Note that the same affinity-purified polyclonal anti-APRIL antibody was used for immunoprecipitations as well as for western blot; precipitated immunoglobulins (>46 and 25 kDa) were developed in western blot as well. An additional control immunoprecipitation of transfectant culture supernatants using non-specific rabbit immunoglobulin is thus shown. (C) An inhibitor for furin convertase, but not for metalloprotease, abrogates APRIL processing. Cells were treated for 5 h with 50 µM chloromethylketone derivative (furin convertase inhibitor) or 10 µM KB8301 (metalloprotease inhibitor) and subsequently analysed. (D) Deletion of amino acids 101–104 abrogates APRIL processing. HEK 293 cells stably transfected with Flag-tagged APRIL, Flag-tagged ΔAPRIL mutant lacking amino acids 101–104 or empty vector (mock), and cell extracts were analysed by western blot employing affinity-purified polyclonal anti-APRIL antibody. (E) Soluble APRIL cannot be immunoprecipitated from culture supernatants of chloromethylketone-treated APRIL transfectants and ΔAPRIL-transfected HEK 293 cells. Immunoprecipitates of culture supernatants derived from APRIL-, ΔAPRIL- and mock-transfected cells were analysed by western blot. (F) Analysis of APRIL processing during maturation. HEK 293T cells were transiently transfected with truncated or mutated APRIL constructs and cell extracts were analysed by western blot using polyclonal anti-APRIL antibody affinity purified on a peptide spanning amino acids 124–146. (G) APRIL digestion by furin in vitro. APRIL and deleted APRIL (ΔAPRIL) were in vitro translated, immunoprecipitated and either mock digested or digested with furin, alone or in the presence of the furin inhibitor chloromethylketone (CMK). Proteins were separated on 15% SDS–polyacrylamide gels.

Fig. 2. APRIL associates with the Golgi apparatus. (A) APRIL is intracellularly localized. APRIL staining is shown in green, DNA staining with Topro-3 in red. (B) APRIL colocalizes with anti-human golgin-97 (CF4) monoclonal antibody, a marker for Golgi apparatus. APRIL staining is shown in green, staining for golgin-97 (CF4) in red. Yellow visualizes the overlap of the two fluorescent antibodies. Images are shown of transiently transfected 293T cells. Similar images were obtained on stably transfected cells. Asterisks indicate non-transfected cells serving as negative controls. Bar, 50 µm.

Fig. 3. Processing of APRIL takes place in the Golgi apparatus. (A) BFA abrogates cleavage of APRIL. HEK 293T cells were transiently transfected with APRIL, and treated for 5 h with BFA (3.5 µM), monensin (3.5 µM), chloromethylketone derivative (CMK; furin convertase inhibitor, 50 µM) or KB8301 (metalloprotease inhibitor, 10 µM) and subsequently analysed by western blot employing affinity-purified polyclonal anti-APRIL antibody. Proteins were separated in 15% SDS–PAGE. (B) Soluble APRIL cannot be immunoprecipitated from culture supernatants of BFA- or monensin-treated APRIL transfectants. Immunoprecipitates of culture supernatants from APRIL-transfected cells treated with BFA or monensin for 5 h were analysed by western blot. Proteins were separated on a 15% SDS–polyacrylamide gel. (C) Treatment with BFA, but not monensin, causes delocalization of APRIL. Cells were incubated with BFA (3.5 µM) or monensin (3.5 µM) for 5 h prior to fixing. Staining for golgin-97 or APRIL is shown in red, DNA staining with SybrGreen in green. Asterisks indicate non-transfected cells serving as negative controls. Bar, 10 µm.

Fig. 4. Secreted APRIL induces additional proliferation of Jurkat cells. Jurkat cells were incubated for indicated time points in the presence of culture supernatant derived from mock-, ΔAPRIL- and APRIL-transfected HEK 293 cells (1:2 dilution with fresh RPMI medium). Proliferation was measured by [³H]thymidine incorporation. Data are the mean ± SEM of triplicate determinations. Proliferation of control treated cells is taken as 100%. Using ANOVA analysis of variance, the following values were obtained: p >0.05 for day 1; p <0.01 for days 3, 5 and 7.