Fig 2: SERBP1-silencing downregulates Ago2 protein levels in SH-SY5Y cells. (A) Representative Western blot of extracts from cells transfected with control siRNA, Ago2 siRNA or SERBP1 siRNA from three independent experiments. Nitrocellulose membranes were hybridized with Ago2 or SERBP1 antibodies, as indicated. The GAPDH signal was used to normalize different samples (B). Fold change of Ago2 and SERBP1 protein levels in siRNA transfected cells relative to control siRNA samples are shown. Data represent the mean of three independent experiments ± standard error (S.E.); * p < 0.05 (t-test).. (C) RT-qPCR analysis of RNA extracted from SH-SY5Y cells transfected with control siRNA, Ago2 siRNA or SERBP1 siRNA. ΔCt values were normalized to TBP level. Fold change of Ago2 and Serbp1 mRNA expression levels relative to control siRNA samples are shown. Individual data points for Ago2 (circles) and Serbp1 (triangles) siRNA transfected cells are indicated. Data represent the mean of three independent experiments ± S.E.; * p < 0.05, ** p < 0.01.

Fig 3: Ago2 interacts with SERBP1. (A) SDS-PAGE stained with Comassie Brilliant Blue of IP with FLAG antibody in SH-SY5Y stably expressing FLAG (lane 1) and FLAG-Ago2 (lane2). Bands corresponding to Ago2-associated proteins are indicated. (B) Cells were co-transfected with Myc-SERBP1 and Flag-Ago2 (lane 1) or with Myc and Flag-Ago2 as the negative control (lane 2) and immunoprecipitation was performed with anti-Myc antibody conjugated beads. Flag-Ago2 was detected by Western blot using anti-FLAG monoclonal antibody.

Fig 4: SERBP1 interacts with KCC2 3′UTR. (A) SH-SY5Y cells were co-transfected with pCDNA3-myc or pCDNA3 myc-SERBP1 together with KCC2 3′UTR. Immunoprecipitates were analyzed by qRT-PCR to determine the levels of KCC2 mRNA in the two different conditions. Data represent the mean of three independent experiments ± S.E.; * p < 0.05. Individual data points for pcDNA3 myc-Serbp1 (squares) transfected cells are shown. (B) Western blotting showing Ago2 and SERBP1 protein levels in the input, eluates and supernatants from cytoplasmic lysates of SH-SY5Y cells (left panels) or hippocampal neurons (right panels) incubated with KCC2 sense or antisense 3′UTR transcripts. Eluate (1) and eluate (2) indicate the first and second elution. (C) Levels of miR-92 bound to KCC2 sense or antisense 3′UTR transcripts were analyzed by qRT-PCR. ΔCt values were normalized to the snU6 level *** p < 0.001.