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The blots were next probed with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and developed using ECL™ (GE Healthcare, Piscataway, NJ).
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Southern blots were probed with the probe DMRP recognizing a 4.2 kb methylated and 2.7 kb unmethylated band [27].
The next day, blots were rinsed and probed with the appropriate horseradish-peroxidase secondary antibody for 1 hour at room temperature in 5% milk in TBST and illuminated with Western Lightening TM Plus-ECL Enhanced Chemiluminescence Substrate assay kit.
To investigate ASAP expression, human multiple-tissue Northen blots were probed with an ASAP cDNA probe.
The blots were then probed with Western Lighting plus-ECL (Perkin Elmer, Inc).
Blots were also probed with U6 probes to normalize loading.
Blots were also probed for actinin (anti-actinin, Sigma).
Duplicate blots were also probed with an anti-6His antibody.
Blots were then probed overnight at 4°C with primary antibodies.
Then, the blots were then probed with ImmunoStar Reagents (Wako).
Blots were also probed for parkin to ensure pulldown.
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