Apb

St A

-167 bp

Fig. 2. Evidence for the expression of aquaporin-9 (AQP9) in ARPE-19 cells by Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma membranes were purified and processed for Western blotting as described by Dibas et al. 2005 (126). (A), a doublet of 37 and 47 KDa is seen in ARPE-19 cells. Total RNA was extracted with Trizol (Life Technology) as described by the manufacturer. In this study, 5 |g of total RNA was reverse transcribed using the iscript kit (Bio-Rad) according to the manufacturer's instructions. Control RT-PCR reactions were performed in the absence of cDNA templates. Polymerase chain reaction (PCR) products were run on agarose gels and bands were cut and sequenced to verify identity. See Tables 2 and 3 for primer sequences and conditions. (B) AQP9 mRNA is expressed in ARPE-19 cells (St: DNA ladder, A: ARPE-19).

Table 2 Gene-specific forward (sense) and Primer name Forward primer (5^3') Aquaporin 9 AGCCACCTCT-

(AQP9) GGTCTTGCTA

p-actin TGTGATGGTGG-

0 0

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