Cell lytic reagent

ICs, TT1 cell line and primary AT2 cells seeded in 6 well plates were lysed with CellLytic reagent (Sigma-Aldrich, Poole, UK) containing a protease inhibitor cocktail P8340 (Sigma-Aldrich, Poole, UK). Protein content was quantified using NanoDrop platform spectrophotometer (Thermo Scientific, Waltham, UK) and adjusted to 50 µg per sample. Proteins were resolved on 10% NuPAGE Bis–Tris gels (Invitrogen, Paisley, UK) and transferred to nitrocellulose membranes using an iBlot 7-min Blotting System (Invitrogen, Paisley, UK). Membranes were blocked for 1 h with 5% bovine serum albumin (BSA) (Sigma-Aldrich, Poole, UK) in TBST buffer (20 mM Tris; 50 mM NaCl; 0.1% Tween-20), washed with TBST trice and incubated with 5% BSA in TBST probed with the primary antibody (Supplementary Table S2 online) at 4 °C overnight. Then, membranes were washed with TBST three times and incubated with 5% BSA in TBST probed with the secondary antibody (Supplementary Table S2 online) for 1 h at room temperature and washed as before. Finally, membranes were exposed to an enhanced chemiluminescence reagent (ECL) Prime (GE Healthcare, Little Chalfont, UK) for 1 min and developed on X-ray films.

AD293 cells were transfected with 100 ng pcDNA3-GPR110 per well of 24 well plate by PEI at the ratio of 1:5. Two days after transfection, cells were lysed by cell lytic reagent (Sigma), proteins in cell lysates were separated in 10% Bis-Tris gels at 170 V for 1 h and then transferred onto polyvinylidene difluoride (PVDF) membranes at 100 V for 1.5 h. The membranes were blocked with 10% milk in TBS-T (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1% Tween-20) at room temperature for 30 min. One of the membranes was incubated at room temperature for 2 h with monoclonal anti-FLAG M2-peroxidase (HRP) antibody (1:5000, Sigma) in TBS-T. The other one was incubated with β-actin mouse mAb (1:10,000, ABclonal) in TBS-T containg 3% milk at room temperature for 2 h, and after being washed with TBS-T, the membrane was incubated for 30 min with HRP goat anti-mouse IgG (1:5000, ABclonal) in TBS-T. After treating with chemiluminescent substrate (Thermo Fisher Scientific), protein bands were detected by iBright CL1000 imaging system (Thermo Fisher Scientific).