Tissues were washed several times with MAB at room temperature, and then developed with BM purple (Roche Diagnostics Corporation) color substrate for 5 16 h.
After incubation and washing, plates were developed by incubation with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Rockford, IL) followed by tetramethylbenzidine substrate (Neogen Corporation, Lexington, KY), then quenched with 0.2 N H2SO4 and read at 450 nm.
HRP activity was detected using 3,3-diaminobendine (DAB) (Nichirei Corporation, Tokyo, Japan) as a substrate.
Horseradish peroxidase chemiluminescent substrate (Millipore Corporation, USA) was used to detect protein blotting by exposure to Kodak BioMax Light films.
Proteins were detected by LuminataTM crescendo western HRP substrate (Millipore corporation, Billerica, MA, USA), using the Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech, San Leandro, CA, USA).
The signals were detected by chemiluminescent substrate kit (Millipore Corporation, Billerica, MA).
Protein bands were detected using an Immobilon Western Chemiluminescent HRP substrate kit (Millipore Corporation, Billerica, MA, USA).
Plates were developed using enhanced K-blue tetramethylbenzidine substrate solution (Neogen Corporation, Ayr, UK), and the reaction was stopped by addition of 1.8 M H2SO4.
For western blot transfer, BioTrace PVDF (polyvinylidene fluoride) membranes (Pall German Laboratory, MI, USA, USA) were used and visualized using the Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate Kit (Millipore Corporation, Billerica, MA, USA) on ImageQuant LAS 500 detection system (GE Healthcare, Upsala, Sweden).
After washing, the membrane was incubated with an appropriate secondary antibody before detection using chemiluminescent horseradish peroxidase substrate (Immobilon Western, Millipore Corporation, MA, USA) (Supplementary Fig. 8).
Immobilon™ Western Chemiluminescent HRP Substrate was from Millipore Corporation (Billerica, MA, USA).
