Figure 4.

Neat1 stabilizes Hbb via inhibiting Hbb ubiquitination

(A) The Neat1 levels were measured in N2a cells transfected with Neat1 GapmeR (∗ ∗p < 0.01, n = 3). (B and C) The Hbb mRNA (B) and protein levels (C) in N2a cells after transfection with Neat1 GapmeR ( ∗p < 0.05, n = 3–6). (D) The Neat1 levels were determined in primary neuronal cells transfected with Neat1 GapmeR for 24 h ( ∗p < 0.05, n = 5). (E and F) The Hbb mRNA (E) and protein levels (F) in primary neuronal cells after transfection with Neat1 GapmeR for 24 h ( ∗p < 0.05, n = 3–4). (G) N2a cells transfected with Neat1 GapmeR were treated with cycloheximide (CHX, 200 μg/mL) for the indicated times, and Hbb protein levels were determined. (H) The protein levels of Hbb in N2a cells transfected with control GapmeR or Neat1 GapmeR for 24 h and treated with MG-132 (5 μM) or DMSO for 16 h were determined by western blot assay. (I) N2a cells transfected with control GapmeR or Neat1 GapmeR were treated with MG-132 (5 μM) for 16 h. Cell lysates were immunoprecipitated with antibodies against Hbb or IgG. The levels of ubiquitination were analyzed by western blot. Bottom: input from cell lysates. IB, immunoblot.