Novex 16 tris glycine gel

Kidneys were homogenized on ice in T-PER (Thermo Fisher) containing Halt phosphatase and protease inhibitor cocktail (Thermo Fisher). Protein samples (50 μg) were then resolved on Novex 16% Tris–Glycine gel (Invitrogen) and transferred onto 0.22 μm PVDF membrane (BioRad) using a wet transfer system for 1 h at 100 mV. Membranes were then incubated with primary antibody (anti-SAA A1, AF2948, 1:1000, R&D) overnight at 4 °C, washed the next day, and incubated with secondary antibody for 1 h. Membranes were then stripped with Restore buffer (Thermo Fisher) for 15 min, then washed, blocked, and stained for actin as described. Band density was quantified using ImageJ (NIH).

SDS-PAGE was performed using a Novex 16% Tris-Glycine gel (Life Technologies/Invitrogen, Carlsbad, CA, USA) and a 15% e-PAGEL mini gel (ATTO Corporation, Tokyo, Japan). The gel was stained with Simply Blue Safe Stain (Invitrogen) or ProteoSilver Stain (Sigma-Aldrich). The apparent molecular weights of the protein bands were estimated by comparison with the SeeBlue Plus2 Pre-Stained Standard (Life Technologies/Invitrogen) or the Novex Sharp Protein Standard (Life Technology/Invitrogen). Duplicate gels were transblotted onto Invitrolon polyvinylidene difluoride (PVDF) membranes (Life Technologies/Invitrogen) and immunostained with porcine amelogenin or TGFBR1 (Abcam, Cambridge, UK) polyclonal antibodies. Immunopositive bands were visualized by 3,3′-diaminodbenzidine (DAB) for amelogenin or enhanced chemiluminescence for TGFBR1. Full images of the blots that were cropped in the main figures are shown in Supplementary Fig. S12.