Structs are given in Supplementary Table 2. Sequence and structural modeling and evaluation. Various sequence alignments had been performed applying Clustal Omega61. Structural alignments have been generated with PyMOL (www.pymol.org) primarily based on crystal structures from the PDB database (1F(IL-12)62, 3DUH (IL-23)28). Missing loops were modelled with Yasara structure (www.yasara.org) using a subsequent steepest descent power minimization. Structures were depicted with PyMOL. Cell culture and transient transfections. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing L-Ala-L-Gln (AQmedia, Sigma-Aldrich) supplemented with ten (vv) fetal bovine serum (Biochrom or Gibco) at 37 and 5 CO2. Medium was complemented having a 1 (vv) antibiotic-antimycotic resolution (25 gml amphotenicin B, ten mgml streptomycin, and ten,000 units of penicillin; Sigma-Aldrich). Transient transfections have been carried out for 24 h either in p35 or p60 poly D-lysine coated dishes (VWR) using GeneCellin (BioCellChallenge) in accordance with the manufacturer’s protocol. IL-23 DNA and IL-12 DNA or empty vector (in absence of IL-12) had been (co-)transfected within a ratio of 1:two for redox-, secretion- and degradation-experiments. 3 micrograms IL-23 DNA had been applied for co-immunoprecipitation experiments. To analyze BiPinteractions, 1 g hamster BiP DNA was co-transfected with IL-23. Biotin-azide Chemical Immunoblotting experiments. For secretion, redox status experiments and knock down experiments with siRNA, cells were transfected for 8 h in p35 dishes, washed twice with PBS then supplemented with 0.five ml fresh medium for one more 16 h. For siRNA experiments cells were transfected with 25 nM siRNA (Thermo Fisher) for 24 h prior to DNA transfection. siRNA was diluted in Opti-MEMTM Reduced Serum Medium and transfected with LipofectamineRNAiMAX Transfection Reagent (Thermo Fisher). For CHX chase assays cells were treated with 50 ml CHX (Sigma-Aldrich) for occasions indicated in the figures ahead of lysis. Protein halflives ( D) were calculated from exponential fits on the curves. To analyze secreted proteins, the medium was centrifuged for five min, 300 g, four . Subsequently, samples were supplemented with 0.1 volumes of 500 mM TrisHCl, pH 7.5, 1.5 M NaCl (and 200 mM NEM in the case of non-reducing SDS-PAGE) and protease inhibitor and centrifuged for 15 min, 20,000 g, four . Before lysis, if indicated, cells have been treated with 10 mM DTT (Sigma-Aldrich) for the last hour or 1 ml Brefeldin A (Sigma-Aldrich) for 2.five h, washed twice in ice cold PBS, supplemented with 20 mM NEM if samples were to be analyzed by non-reducing SDS-PAGE. Cell lysis was carried out in RIPA buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1.0 Nonidet P40 substitute, 0.five sodium deoxycholate, 0.1 SDS, 1x Roche complete Protease Inhibitor wo EDTA; Roche Diagnostics) or Triton lysis buffer within the case of coimmunoprecipitation experiments (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 Triton X-100, 1x Roche (S,R)-Noscapine (hydrochloride) Biological Activity comprehensive protease inhibitor wo EDTA, supplemented with 10 Uml Apyrase for BiP interaction research (Sigma-Aldrich) or 20 mM NEM (Sigma) for PDI and Erp44 co-IPs). Samples have been supplemented with 0.2 volumes of 5x Laemmli containing either -Me for minimizing SDS-PAGE or 100 mM NEM for non-reducing SDS-PAGE. Deglycosylation assays with Endo H (New England Biolabs), PNGase F (SERVA) or a mix of O-glycosidase and 2,six,8 Neuraminidase (New England Biolabs, cleavage of O-glycosylations) were performed according to the manufacturers’ protocols.