Mouse retina cDNA was prepared as described [135] (link). vps26A was PCR-amplified using primers 5′-GAGTTTTCTTGGAGGCTTTTTTGGTCC-3′ and 5′-TTACATCTCAGGCTGCTCCGCAGAGG-3′ and a 30 ng cDNA template. The PCR product was cloned into pBluescript by blunt-end ligation and verified by sequencing. DNA probes were generated from gel-purified insert (excised with EcoRI and HindIII) by random-prime labeling with [α-32P]dCTP using the DECAprimeII kit (Ambion). Unincorporated nucleotides were removed with a MicroBio-Spin 30 column (Bio-Rad).
For Northern blot analysis, total RNA was prepared from retinas of 3–4-mo-old CD-1 mice by homogenization in TRI reagent (Ambion) using a motorized pestle and passage through a 20-gauge needle, extraction with 1-bromo-3-chloro-propane, and purification with the RNeasy kit (Qiagen). We resolved 10 µg of total RNA on a formaldehyde-agarose gel and transferred it to a Hybond-N+ nylon membrane (GE Healthcare) by capillary transfer. Membranes were prehybridized in ULTRAhyb (Ambion) for 1 h, then incubated at 42°C overnight with a ∼2.5×107 cpm denatured probe diluted in ULTRAhyb. Blots were washed several times in 2× SSC+0.1% SDS and several times in 0.1× SSC+0.1% SDS. Washes were performed at 42–45°C. For imaging, blots were exposed to a storage phosphor screen and scanned on a Typhoon (GE Healthcare).
For Northern blot analysis, total RNA was prepared from retinas of 3–4-mo-old CD-1 mice by homogenization in TRI reagent (Ambion) using a motorized pestle and passage through a 20-gauge needle, extraction with 1-bromo-3-chloro-propane, and purification with the RNeasy kit (Qiagen). We resolved 10 µg of total RNA on a formaldehyde-agarose gel and transferred it to a Hybond-N+ nylon membrane (GE Healthcare) by capillary transfer. Membranes were prehybridized in ULTRAhyb (Ambion) for 1 h, then incubated at 42°C overnight with a ∼2.5×107 cpm denatured probe diluted in ULTRAhyb. Blots were washed several times in 2× SSC+0.1% SDS and several times in 0.1× SSC+0.1% SDS. Washes were performed at 42–45°C. For imaging, blots were exposed to a storage phosphor screen and scanned on a Typhoon (GE Healthcare).
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