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. 2017 Jun;23(6):910–926. doi: 10.1261/rna.060640.117

FIGURE 6.

FIGURE 6.

SKIV2L2 binds to and modulates the half-life of replication dependent histone mRNAs. (A) Western blot to detect immunoprecipitated SKIV2L2 in N2A cells. Western blots to detect SKIV2L2 were performed on the following samples from N2A cells. (Lane 1) Whole-cell extract incubated with anti-goat IgG. (Lane 2) Immunoprecipitate with anti-goat IgG. (Lane 3) Whole-cell extract incubated with anti-SKIV2L2. (Lane 4) Immunoprecipitate with SKIV2L2. The top band indicates the position of SKIV2L2, and the lower band represents the heavy chain from the antibody used for immunoprecipitation. (B) Western blot to detect immunoprecipitated SKIV2L2 in P19 cells. Western blots to detect SKIV2L2 were performed in P19 cells as stated above. (Lane 1) Whole-cell extract incubated with anti-goat IgG. (Lane 2) Immunoprecipitate with anti-goat IgG. (Lane 3) Whole-cell extract incubated with anti-SKIV2L2. (Lane 4) Immunoprecipitate with SKIV2L2. (C) qRT-PCR of histone RNAs bound to immunoprecipitated SKIV2L2 in N2A cells. Proteins were extracted from N2A cells irradiated with UV light. SKIV2L2 was immunoprecipitated with its cross-linked RNAs using anti-SKIV2L2, with anti-goat used as a negative control. RNA was isolated from both anti-SKIV2L2 and anti-goat immunoprecipitates. Using qRT-PCR, RNA abundance levels of histone mRNAs immunoprecipitated in either sample were calculated using ΔCq values, and normalized to the amplification of Skiv2l2 mRNA, which was unbound in both samples (error bars represent ±SD for n = 3). Immunoprecipitated retro-Phgdh represents an RNA precipitated equally by anti-goat and anti-SKIV2L2. (D) qRT-PCR of histone RNAs bound to immunoprecipitation SKIV2L2 in P19 cells. Performed as stated in C on immunoprecipitate from P19 cells. (E) Northern blot demonstrating H4 mRNA turnover following Dactinomycin treatment. Following transfection with control or Skiv2l2 siRNA, RNA was extracted at 0, 1, 2, 3, and 4 h following application of Dactinomycin. Northern blotting detected H4 mRNA, which was quantified and normalized to ribosomal RNA levels to calculate half-life.