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Blots were again washed three times with TBS and then developed using enhanced chemiluminescence (ECL) kit.
The blots were again washed in PBS Tween and processed for development using chemiluminescence reagent (Millipore, USA).
Blots were again washed with TBS-T and the signal was detected using chemiluminescence reagent (Millipore, Bedford, MA, USA).
The blots were again washed in TBS for 40 min prior to secondary antibody incubation with either, rabbit anti-mouse IgG, and goat anti-rabbit IgG conjugated to horseradish peroxidease (0.2 µg/mL) for 1 hr.
The blots were again washed in TBS-T.
The blots were again washed, developed by chemiluminescence, and exposed to radiographic film.
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The blot was again washed with sodium phosphate buffer and developed with NBT/BCIP.
The blot was again washed three times with TBS before being exposed to the SuperSignal West Dura Extented Duration substrate (Pierce Biotechnology, Rockford, IL).
The blot was again washed 3× with TBST for 30 minute at RT followed by chemiluminescent detection (Pierce Super-Signal dura) on HyBlot CL autoradiography film (Denville Scientific).
The membranes were again washed three times with the buffer, and the proteins were visualized using ECLTM western blotting detection reagents (GE Healthcare).
Plates were again washed extensively.
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