Skip to main content
. Author manuscript; available in PMC: 2010 Jul 10.
Published in final edited form as: J Neurochem. 2007 Aug 30;103(4):1491–1505. doi: 10.1111/j.1471-4159.2007.04858.x

Fig. 3.

Fig. 3

CD44 deoxyribozyme specifically down-regulates CD44 expression in RGCs. (a–c) Confocal images of RGCs growing for 24 h on different substrates as marked were treated with biotin-labeled deoxyribozyme to CD44 in a concentration of 8 µmol/L. Red shows the labeled deoxyribozyme localized in the cytoplasm; green is β-tubulin immunostaining. In each image, cross-sections (lower or right-hand sides of the image) demonstrate the accumulation of the deoxyribozyme in the cytoplasm (yellow/orange). (d) RT-PCR for CD44 (40 cycles) with RNA from RGCs after 24 h in culture. Reduction of CD44 mRNA after deoxyribozyme treatment against CD44 (CD44AS), as compared to control deoxyribozyme treatment (CD44MB), or to untreated cultures, was observed. (e) RT-PCR as in (d) for β-actin (25 cycles); bands demonstrate similar RNA template sample concentrations, and specificity of CD44 down-regulation from (d); MWM, molecular weight marker. (f–h) Immunostainings of RGCs to CD44 (red channel) and β-tubulin (green channel) after 24 h in culture demonstrate a reduction of CD44 after deoxyribozyme treatment to CD44 (f), compared to control deoxyribozyme treatment (g) and untreated (h) cultures. Note the strong positive CD44 staining at the short but multiple neurites of (g) and at the end-feet of the axonal growth cone in (h). (i and j) Immunoprecipitation of CD44 protein (i) or β-tubulin (j) from RGCs after treatment with 8 µmol/L of a deoxyribozyme targeting CD44 mRNA (CD33AS), 8 µmol/L control deoxyribozyme (CD44MB), or left untreated. U937 is a human leukemia monocyte lymphoma cell line, which was used as positive control, based on its known high expression of CD44. (k) Shows a Coomassie stained 4–15% SDS gel, which was loaded with crude protein extract from RGCs treated with 8 µmol/L of deoxyribozyme to CD44 (CD44AS), control deoxyribozyme (CD44MB), left untreated or as a positive control U937 cell extract. Note that the overall protein bands are equal in each lane which demonstrates that deoxyribozyme treatment has no general effect on the RGCs.