PMC

(A) Immunoblots showing reactivity of LruB-antiserum (1∶400) with recombinant human β-crystallin B2(1µg). (B) Pre-immune serum (1∶400) did not react with β-crystallin B2. Molecular mass of recombinant β-crystallin B2 is 50.4 kDa. Molecular mass markers are indicated in kilodaltons.

(A) Immunoblot showing reactivity of LruA-specific antiserum (1∶400) with recombinant human alpha-crystallin B (1µg). (B) Pre-immune serum (1∶400) did not react with this protein. (C) Immunoblots showing reactivity of LruA-antiserum (1∶400), but not the pre-immunization serum (D), with purified vimentin (1µg). Molecular mass markers are indicated in kilodaltons.

(A) Photomicrographs showing uniform homogeneous fluorescence in a section of equine lens incubated with LruA-antiserum (1∶100) but not with preserum (B) (×100). (C) Photomicrographs showing a positive fluorescence in a frozen section of equine retina incubated with LruB-antiserum (1∶100) but not with pre-immunization serum (D) (×100). Inset (×400).

(A) Lens extract separated on a polyacrylamide gel stained with the fluorescent dye SYPRO-Ruby. (B) Lens proteins transferred from a second gel to nitrocellulose membrane and blotted with LruA-antiserum. The arrowheads indicate the protein spots excised from the stained gel for analysis by mass spectrometry. (C) Retinal extract separated on a polyacrylamide gel stained with SYPRO-Ruby. (D) Retinal proteins transferred from a second gel to nitrocellulose membrane and blotted with LruB-antiserum. Three protein spots (numbered 3, 4 and 5) were excised from the stained gel for analysis by mass spectrometry. Results of mass spectrometric analyses are tabulated in .

ELISA results showing significantly higher antibody levels of alpha-crystallin B (A), vimentin (B) and β-crystallin B2-antibodies (C) in uveitic eye fluids. ELISA plate wells were coated with 200 ng of each eye protein. After blocking, wells were sequentially incubated with uveitic or normal eye fluids diluted 1∶100 and HRP-conjugated protein G (1∶5000). Plates were developed using 3,3′,5,5′-tetramethyl benzidine substrate solution and absorbance measured at 450 nm. Presented data is a representative of at least two independent experiments with 3 or more repeats. Error bars indicate standard deviation.