Research Paper Volume 15, Issue 19 pp 10705—10731

Figure 9. cGlra2 acts as a miRNA sponge in RGCs. (A, B) FISH assays (A) and qRT-PCR assays (B) were conducted to detect the cytoplasmic and nucleus expression of cGlra2 in RGCs. Scale bar: 20 μm. (C) The cytoplasmic fractions of RGCs were immunoprecipitated by Ago2 antibody or IgG. The amount of cGlra2 in RGCs were determined by qRT-PCRs (n = 4, P < 0.05). (D) The entire cGlra2 sequence was cloned into pGL3 Luciferase Reporter to build Luc-cGlra2 vector. RGCs were co-transfected Luc-cGlra2 with different miRNA mimics. Luciferase activity was determined by the dual luciferase assay following 48-h transfection (n = 4, P < 0.05). (E) The 3’-end biotinylated miR-144 or miR-23 were transfected into RGCs. Following streptavidin capture, the amount of cGlra2 or cZNF609 in bound fractions were determined by qRT-PCR assays. Relative immunoprecipitate (IP)/input ratios were plotted (n = 4, P < 0.05). (F) RGCs were transfected with miR-144 mimics, scramble (Scr) miRNA mimics, or left untreated (Ctrl) for 24 h. qRT-PCRs were conducted to detect the levels of BCL211 expression (n = 4, ^*P < 0.05). (G) RGCs were co-transfected LUC-BCL211 with or without miR-144 mimics or Scr miRNA mimics. Luciferase activity was determined by the dual luciferase assays at 24 h post transfection (n = 4, ^*P < 0.05).