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Subsequently, the blots proceeded through a second round of immunodetection by agitating for 45 min at 50C in stripping buffer (2% SDS, 0.8% -mercaptoethanol (ME), and 62.5 mM Tris-HCl, pH 6.8) 32,56,62 followed by re-probing with HRP-labeled anti-HGF N -terminal domain mAb (1:2500 dilution in CanGetSignal solution 1) to monitor HGF -chain amount on the blots as loading controls