Of interest is the truth that in both cells expression was markedly higher than for p16INK4A. (CST Ab; B) and Santa-Cruz (sc Ab; C) PF6-AM elicited weaker labelling of inflammatory cells (observe arrows). Level pub?=?30 m.(TIF) pone.0075067.s002.tif (1.8M) GUID:?82E78079-2237-44B4-9F3F-E4C590622F0A Number S3: Evaluation of p16INK4A antibodies, and p16INK4A expression in rat cells, by Western immunoblotting. (A) Evaluation of four p16INK4A antibodies, from ProteinTech (PT), Sigma, Santa-Cruz (sc) and Abnova, by Western blotting, using GST-tagged, full length, recombinant, human being p16INK4A protein (rp16INK4A). For each antibody tested, molecular excess weight markers were used to determine size of recognized gel products (A; lane M). Lane 1, 100 ng of rp16INK4A; Lane 2, 10 ng rp16INK4A. Solitary bands of the expected molecular weights (including GST-tag) are apparent for each of the tested antibodies except the Abnova one. (B) Rat mind cortex (lane 1), liver (lane 2), optic nerve (lane 3) and retina (lane 4) samples probed for p16INK4A protein with the PT, Sigma, sc and Abnova antibodies. None of the antibodies detect proteins at the correct PF6-AM molecular mass for p16INK4A in any rat cells sample. Labelling for -actin (house-keeping gene product) is also demonstrated. (C) The Sigma and PT antibodies were further tested against retina (lanes 1C3) and optic nerve (lanes 4C6) samples from three different rats. The presence of p16INK4A protein is not detectable in any sample.(TIF) pone.0075067.s003.tif (925K) GUID:?CDB7F22F-EA05-4902-BFF6-8D49BCCAC173 PF6-AM Figure S4: Evaluation of antibodies directed against p16INK4A in cervical adenocarcinoma. In formalin-fixed, paraffin-embedded cells sections, incubation with either the Santa-Cruz (sc; A), Proteintech (PT; B) or Sigma (C) antibodies identifies so-called block or diffuse immunolabelling of ductal cells with PF6-AM a high signal-to-background, as compared to adjacent cells (arrow). The staining intensity is very best for the sc Ab and weakest for the Sigma antibody. The Abnova (D) antibody only inconsistently labels irregular cells and, additionally, generates nuclear labelling of adjacent cells (reddish asterisk). Further examination of the Abnova antibody in cervical intraepithelial PF6-AM neoplasia reveals the pattern of immunolabelling again differs from that acquired with the sc Ab (E) featuring weaker staining of the epithelium and strong nuclear localisation of numerous cells residing in the stromal cells (F; reddish asterisk). Level pub?=?30 m.(TIF) pone.0075067.s004.tif (3.6M) GUID:?58734EDC-BFA7-4136-9B02-3641C1F08B5A Number S5: Representative images of p16INK4A immunolabelling in human being ocular cells. In formalin-fixed, paraffin-embedded human being eyes, no positive labelling for p16INK4A is definitely discernible in the corneal epithelium, trabecular meshwork (TM)/Schlemms canal (SC), or retina, irrespective of whether the Sigma (ACC) OR Proteintech (DCF) antibody is used. Level pub: A, B, D, E, G, H?=?15 m; C, F, I?=?30 m. GCL, ganglion cell coating; INL, inner nuclear coating.(TIF) pone.0075067.s005.tif (3.2M) GUID:?3A76C747-9C73-4470-AFE4-FA32DE0CDC3F Number S6: Representative images of p16INK4A immunolabelling in rat retina and optic nerve. In formalin-fixed, paraffin-embedded rat retina (ACC) and optic nerve (DCF), no unambiguous, positive labelling for p16INK4A is definitely discernible either in retina or in the optic nerve using the Sigma (A, D) or Proteintech (B, E) antibodies. In contrast, the Abnova antibody robustly labels all IL7R antibody cell nuclei within the eye, as highlighted in the offered images of retina (C) and optic nerve (F). Level pub: ACD?=?30 m; ECH?=?15 m. GCL, ganglion cell coating; INL, inner nuclear coating; ONL, outer nuclear coating.(TIF) pone.0075067.s006.tif (3.3M) GUID:?88245911-A703-4C8F-8453-5D7B0736456E Number S7: Representative images of p14ARF immunolabelling using the antibody from CST. In formalin-fixed, paraffin-embedded sections of cervical intraepithelial neoplasia, p14ARF manifestation is.