FIGURE 2. αA-crystallin is expressed in CCD cells and co-immunoprecipitates with ENaC α subunit.

αA-crystallin is expressed in mouse kidney tissue and cultured CCD cells (A and B). One microgram of total RNA isolated from native mouse kidney tissue (A) or cultured CCD cells (B) was reverse transcribed using oligo dT (dT) or random hexamers (dN6, CCD cells only) as primers (n = 2). Negative controls were performed in reactions lacking reverse transcriptase (No RT). Predicted reverse transcription-PCR product sizes were 224 bp for αA-crystallin outer primers, 175 bp for αA-crystallin nested primers, and 174 bp for β-actin primers. Note that only nested primers yielded a strong signal of the expected size. C, cultured CCD cell lysates and cytoplasmic extracts were immunoblotted (IB) with mouse anti-αA-crystallin (n = 2). Purified bovine αA-crystallin protein was added as indicated. MDCK cell lysates were immunoprecipitated and immunoblotted with anti-αA-crystallin (n = 3). αA-crystallin is indicated with an arrow. D, ENaC α subunit and αA-crystallin co-immunoprecipitate. MDCK cell extracts were transfected with vectors engineered for the expression of αA-crystallin, ENaC α^HA or α^V5 subunit, and ENaC β and γ subunits as indicated. Extracts were immunoprecipitated with the indicated amounts of either anti-HA antibody or anti-αA-crystallin antibody. IB: αA-crystallin, n = 4; IB: HA, n = 2. αA-crystallin is indicated with an arrow. The furin-cleaved (65 kDa, ►) and uncleaved (95 kDa, >) ENaC α subunit products are also indicated.