Axiocam 305 colour camera

IHC was performed on 7 μm sections of murine spleens as described in Ref. 62. Imaging was performed on a Zeiss Axioplan 2 microscope using a 10× objective (Zeiss, Oberkochen, Gemany) and the Axiocam 305 colour camera (Zeiss). Pictures were generally processed with the zeiss zen blue 2.5 lite software (Zeiss).

The following devices and softwares were used: (1) Zeiss Axiolab 5 microscope with Axiocam 305 colour camera for immunohistochemistry (Zeiss ZEN v3.7), (2) Leica TCS SP8 confocal laser scanning microscope (LAS X v.5.7.23225), (3) Zeiss Supra 40 Field Emission Scanning Electron Microscope to acquire transmission electron microscope (Zeiss SmartSEM v6.0) and 3D Modeling (3DMOD v4.9.10), (4) XF96 and X24 Seahorse analyzers (Agilent Technologies) to collect OCRs (WAVE Pro v10.0.1.84; v2.6.1.56, respectively), (5) Synergy HTX (BioTek) multi-mode plate reader (Gen5 v3.12), (6) StepOnePlus Real-Time PCR System (Applied Biosystems) for mRNA expression (StepOne v2.3), (7) Microsoft Excel v16.48, Microsoft Word v16.48, Microsoft PowerPoint v16.47, (8) Prism v8.4.3, (9) Endnote X9.3.3 and (10) ImageJ v2.0.0-rc-69/1.52p. Enrichment map was generated in Cytoscape v3.8.1, Enrichment map plugin v3.3.0. Handling and analyses of proteomics data were performed via Python v.3.7.4 and Scientific Python Stack: SciPy v.1.3.1, NumPy v.1.17.2 and Matplotlib v.3.1.1. Phosphosites showing significant regulation between groups were used to predict the kinase responsible for their catalysis using the iGPS software.

Flag was detected using a Mouse on Mouse (MOM) ImmPRESS HRP (Peroxidase) Polymer Kit (Vector Laboratories, MP-2400). Paraffin-embedded livers were cut into 5 μm sections, deparaffinated in xylene and rehydrated in a series of graded alcohols and water. For antigen unmasking, sections were incubated in citrate-based antigen unmasking solution (pH 6.0; Vector Laboratories, H-3300) at high temperature for 20 min. After blocking in BLOXALL Endogenous Blocking Solution (Vector Laboratories, SP-6000) for 10 min and a 1 h incubation in MOM Mouse IgG Blocking Reagent, sections were stained with mouse monoclonal anti-DYKDDDDK Tag antibody (1:100; Cell Signaling Technology, 8146) in 2.5% normal horse serum MOM solution overnight at 4 °C. DYKDDDDK signal was revealed by incubation with MOM ImmPRESS Reagent for 10 min and enhanced with ImmPACT DAB EqV Peroxidase (HRP) Substrate (Vector laboratories, SK-4103) for 1 min. Sections were counterstained with haematoxylin, dehydrated, mounted with Permount mounting medium (Fisher, SP15) and imaged in a Zeiss Axiolab 5 microscope/Axiocam 305 colour camera (Carl Zeiss Microscopy). Quantification of Flag percentage area was performed as described⁷⁸ .