Thsd7a

Paraffin sections of 5 μm in thickness were subjected to routine rehydration and antigen retrieval before being incubated with antibody against cytokeratin8 (CK8,1:300; Novus Biologicals, Novus, USA) or THSD7A (1:50; R&D, SANTA, USA). The sections were further incubated with secondary antibody conjugated with horseradish peroxidase (Zhongshan Goldenbridge, Beijing, China) and were visualized with diaminobenzidine (Zhongshan Goldenbridge, Beijing, China) as a substrate.

PLA2R, CysR domain, THSD7A (R&D Systems) and TSR1 protein samples (1 μg per lane) were resolved using 4–12% BisTris gradient gels with the MES buffer system, transferred to nitrocellulose membranes and blotted using patient sera (1:100 dilution) followed by incubation with Alexa Fluor® 680-AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (min X Bov,Hrs,Ms Sr Prot) (1:10,000 dilution; Jackson Labs). Proteins were also analyzed using Moab 20-2-6, a monoclonal anti-PLA2R mouse antibody raised against a PLA2R fragment (see 2.4.1 for further details) at dilution 1:5000. Protein bands were visualized using an Odyssey Imaging System (LI-COR) at 700 nm and the intensity settings were kept constant for all blots allowing comparison. For slot blotting, 1 μg of proteins or peptides (P28mer aa38–65 and T28mer aa75-102 synthesized by ProImmune Ltd, Oxford, UK) were blotted directly onto nitrocellulose membrane and vacuum applied. For the reducing condition, samples were diluted in 10 mM Tris pH 8 then reduced and alkylated by sequential incubation with 50 mM DTT for 1 h at 37 °C and then with 125 mM iodoacetamide for 20 min in the dark. The reduced samples along with unreduced were blotted as described above.