Ab39162

Alphafold was used to predict an interaction between iRhom2 and HLA, as it was previously used to model the iRhom2/ADAM17 interaction [29 (link)]. For the co-immunoprecipitation of iRhom2 and HLA, 2.2 × 10⁶ cells were seeded in a 10 cm dish. Then, 10 µg of pcDNA3.1 plasmid containing HA tagged iRhom2, HA tagged inactive signal peptide peptidase-like 2 protease b (Sppl2b-D/A) or empty plasmid were transfected with 30 µl of Lipofectamine 3000 (Invitrogen). After 48 h, cells were lysed in 1 ml of lysis buffer (50 mm Hepes pH 7.4, 150 mm NaCl, 5 mm EGTA, 1% Glycerol, 1% Triton X100, 1.5 mm MgCl₂, 10 mm 1,10-Phenanthroline) supplemented with complete protease inhibitor (Roche, Basel, CH). Cell lysates were cleared by centrifugation at 16,000 g for 20 min at 4 °C. 500 µl of cleared lysates were applied to agarose beads coupled with anti-HA antibodies (A2095, Sigma–Aldrich, US) and incubated o/n at 4 °C. Then, beads were washed 6 times in lysis buffer and incubated with 20 µl of Laemmli buffer at 65 °C for 20 min. Eluted proteins were loaded onto an acrylamide gel, separated by SDS-PAGE electrophoresis and then analysed by Western blotting using following antibodies: anti-ADAM17 (ab39162, Abcam, Cambridge, UK), anti-HA [HA7] (Sigma-Aldrich, St. Louis, MO, US), anti-HLA,ABC [EMR8-5] (ab70328, Abcam, Cambridge, UK) and anti-GAPDH (Code 5174, Cell Signaling, Danvers, Massachussets, US).

The cell monolayer was washed with ice‐cold phosphate‐buffered saline (PBS), harvested in 50‐200 μL lysis buffer. The lysates were sonicated for 4 seconds and separated by centrifugation at 14 000 g for 15 min at 4°C. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Sigma, USA). Lysates containing equal amounts of proteins were separated on a 10% SDS‐PAGE and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% bovine serum albumin (BSA) (Sigma‐Aldrich) in TBST for 3 hours at room temperature. The blots were probed with primary antibodies at 4°C overnight. The above primary antibodies were used at a 1:1000 dilution, the loading control anti‐β‐actin at a 1:4000 dilution and the secondary antibody at a 1:4000 dilution. The membranes were probed using the following primary antibodies: anti‐β‐actin (Proteintech, USA), anti‐ADAM17 (Abcam ab39162, UK), anti‐EGFR (Abcam ab52894, UK) and anti‐p‐EGFR (Abcam ab40815, UK). The images were quantified with the Quantity One 4.1 software (Bio‐Rad, USA).