Evaluation of p19^ARF antibodies, and p19^ARF expression in rat retina and optic nerve.

<p>(A) Evaluation of two p19^ARF antibodies, from Upstate and Santa-Cruz (sc), by Western immunoblotting using full length, recombinant rat p19ARF protein (r P19^ARF). For each antibody tested, molecular weight markers were used to determine size of detected gel products (A, C; lane M). Lane 1, 100 ng of r P19^ARF; Lane 2, 10 ng rP19^ARF. Single bands of the correct molecular mass are detectable by the Upstate antibody, but not by the Santa Cruz antibody. (B) Rat brain cortex (lane 1), liver (lane 2), optic nerve (lane 3) and retina (lane 4) samples probed for p19^ARF protein with the Upstate and sc antibodies. The Upstate antibody does not recognize any proteins at the correct molecular mass, but the sc antibody recognises a 19 kD protein in the optic nerve sample. Labelling for β-actin (house-keeping gene product) is also shown. (C) Further testing of both antibodies against retina (lanes 1–3) and optic nerve (lanes 4–6) samples obtained from three different rats reveals that the Upstate antibody fails to detect a band at 19 kD in any sample, but detects a protein of approximately 38 kD in the retinal, and a protein of 48 kD in the optic nerve, samples. The sc antibody recognises a 19 kD protein in each optic nerve sample but not in the retinal samples. (D) Representative images of p19ARF immunolabelling using the Upstate p19^ARF antibody in the rat retina and optic nerve. No nuclear staining is detectable in cells of the retina or optic nerve; however, robust labelling of axons in the myelinated, but not unmyelinated, optic nerve is apparent. This is most clearly shown in the image taken of the optic nerve head (ONH). Scale bar = 30 µm. GCL, ganglion cell layer; INL, inner nuclear layer.