Af5208

HEK293 cells (ATCC CRL‐1573) were grown under standard conditions and transfected with FLAG (DDK)‐tagged plasmids expressing human or mouse PNPLA2 or PNPLA3, or VHH‐FLAG‐Halo (Table S1). Cells were transfected using Fugene HD (Promega) according to the instructions from the vendor. After 48 hours, cells were lysed and western blot experiments performed as detailed in the Supporting Materials and Methods. The following primary antibodies were used: sheep polyclonal anti‐human PNPLA3 (1:1000, AF5208; R&D Systems, Minneapolis, MN, USA), mouse monoclonal anti‐FLAG M2 (1:1000, F1804; Sigma Aldrich, USA), and rabbit polyclonal anti‐alpha tubulin (1:200, ab4074; Abcam, UK). Membranes were washed and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies anti‐rabbit (1:3000, 7074S; Cell Signaling Technologies, Inc, USA), anti‐mouse (1:3000, 7076S; Cell Signaling Technologies), or anti‐sheep (1:2000, ab6900; Abcam). Western blots were visualized with chemiluminescent HRP substrate (WBLUR0500; Millipore, USA), using the ChemiDoc MP imaging system (Bio‐Rad, USA).

FFPE sections were immunohistochemically stained for PNPLA3 (AF5208; R&D Systems), dilution 1:750 (mouse) and 1:300 (human), using an automated Ventana Ultra system (Ventana Medical Systems, Inc, Roche Group, USA).
The deparaffination and pretreatment were performed in the Ventana system as described in the Supporting Materials and Methods. After staining, slides were scanned into a slide scanner (Pannoramic Scan II; 3DHISTECH Ltd, Budapest, Hungary). Image analysis was performed on digital images using Visiopharm Integrator System software (version 2020.03.0.7300; Visiopharm, Hørsholm, Denmark) (Figure S1C–F).
In the PNPLA3 IHC‐stained slides, the diaminobenzidine‐stained area and the total section area were detected by threshold and machine learning in the Visiopharm image analysis system. The PNPLA3‐positive area was quantified and expressed as a fraction of the total section area excluding blood vessels (i.e., the reported PNPLA3 levels are semiquantitative). Artefacts such as folds in the sections, were removed before performing measurements.