Reducing agent

SDS-PAGE was performed using precast Bis-Tris 12% polyacrylamide gels and an electrophoresis system (Invitrogen, Mount Waverley, Australia). Centrifugal supernatants were diluted (1:25) with deionized water and mixed (10 µl) with 5 µl of LDS (Sigma Aldrich, UK), 2 µl of reducing agent (Sigma Aldrich, UK) and 5 µl of β-mercaptoethanol (Bio-Rad Laborato-ries Ltd., UK). Each of the non-heated reference samples was diluted in a similar way and the same amount of reagents was added. All samples were heated at 100 °C for 3 min prior to loading. The gels were run at 115 V for 90 min and a solution containing 0.025% w/v Coomassie (Bio-Rad Laboratories Ltd., Watford, UK), 40% w/w methanol and 7.5% w/w acetic acid was used for staining for 90 min and a 7.5% w/w acetic acid solution was used for destaining the gels overnight. Gels were visualized using a Fuji Film Intelligent Dark Box II with Fuji Film LAS-3000 V2.2 software (Brookvale, Australia). The proteins were identified by comparison to the molecular weight (Mw 10-250 kDa) of protein standards (Precision Plus, Kaleidoscope, BioRad, Australia).