Polyclonal swine anti rabbit immunoglobulins hrp

For Hnf4g expression in WT and KO organoids: Organoid pellets were resuspended in 100 μl whole cell lysis buffer per well of a 6‐well plate (150 mM NaCl, 50 mM Tris pH 8, 10% glycerol, 1% NP‐40, 1 mM DTT, 1× CPI). Extracts were rotated for 1.5 h at 4°C followed by seven cycles of 30 s on and 30 s off sonication (Biorupter Pico, Diagenode). Afterward, the samples were spun at 16,200 × g for 5 min at room temperature and the supernatant was stored at −80°C. Samples were loaded on an 8% SDS–PAGE gel and afterward transferred to a nitrocellulose membrane. Membranes were incubated with primary antibody in 5% milk/TBST overnight at 4°C [anti‐beta actin 1:5,000 (Abcam, ab16039), and anti‐HNF4G 1:500 (Sigma, HPA005438)]. Afterward, membranes were incubated with secondary antibody [Polyclonal Swine Anti‐Rabbit Immunoglobulins/HRP 1:3,000 (Dako, P0399)] and imaged using chemiluminescent substrate (Thermo Fisher, 34580).
For mitochondrial ETC complexes: Organoids were washed once and Matrigel was mechanically removed in cold PBS. Total proteins were collected by direct lysis of organoids in Laemmli sample buffer. Proteins were run in SDS–PAGE and transferred to Polyscreen PVDF transfer membranes (PerkinElmer). Antibodies: Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam) and anti‐vinculin (V9131, Sigma).

For analysis of IRF5 phosphorylation, DCs were stimulated as indicated in 6-well plates (1,250,000–2,000,000 cell per well) (Costar) for 30 min. Cells were gently scraped and collected in cold PBS. After washing, cells were lysed on ice for 10 min using RIPA lysis buffy (Cell signaling) supplemented with protease and phosphatase inhibitors (both from Roche). Lysates were briefly sonificiated for 10 s at 30% and centrifuged for 10 min at 14,000 × g. BCA assay was performed (Thermo Scientific) and samples were boiled with 4x Laemmli Sample Buffer (Bio-Rad) for 15 min at 95°C. Cell lysates were run on a 4–12% Bis-Tris protein gel (Invitrogen) using MES-running buffer (Invitrogen). Proteins were transferred to a PVDF membrane (GE healthcare) using transfer buffer (Invitrogen) and blocked with 2% milk (Bio-Rad) afterwards. Membrane was incubated in TBS Tween o/n at 4°C with indicated antibodies: Phospho-IRF5 (Ser437) polyclonal antibody (1:1000) (Thermo Scientific), IRF5 (1:1000) (E1N9G) rabbit mAb (Cell Signaling), or Actin antibody (I-19) (1:2000) (Santa Cruz). Afterwards membrane washed with TBS Tween and incubated for 1 h at room temperature with polyclonal swine anti-rabbit immunoglobulins HRP (1:3000) (Dako).