Ab27250

We prepared cell lysates using a buffer (RIPA Lysis and Extraction Buffer 89900, Thermo Fisher Scientific, Waltham, MA, USA). Proteins (30 μg) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane that was blocked with 5% skim milk, and then incubated with an anti-ERC2 antibody (ab27250, Abcam, 1:500), anti-MAFB antibody (#41019, Cell Signalling Technologies, 1:1000) an anti-ACTB antibody (#4970, Cell Signalling Technologies 1:1000) in 5% skim milk. An anti-rabbit IgG conjugated to horseradish peroxidase (#7074, Cell Signalling Technologies, 1:1000) was used as the secondary antibody in Tween 20/100 mM NaCl/10mM Tris-HCL (1:1000 dilution).

For immunohistochemical analysis, after deparaffinization and rehydration, sections were digested with 500 U/mL testicular hyaluronidase for 30 min at 37 °C. Endogenous peroxidase activity was inhibited with 3% H₂O₂ containing 0.1% sodium azide for 5 min at room temperature. Sections were incubated overnight at 4 °C in a humidity chamber with rabbit polyclonal antibodies to the ERC2 (ab27250, Abcam, Cambridge, UK, 1:500) or rabbit monoclonal antibodies to MAFB (#41019, Cell Signalling Technologies, Danvers, MA, USA, 1:1000). Immunostaining reactions were performed using an Envision™+ Kit (K4007, Agilent Technologies, Santa Clara, CA, USA), followed by staining using 3,3′-diaminobenzidine (DAB) reagent.