Journal: Molecular Biology of the Cell

Article Title: SCYL1BP1 modulates neurite outgrowth and regeneration by regulating the Mdm2/p53 pathway

doi: 10.1091/mbc.E12-05-0362

Figure Lengend Snippet: SCYL1BP1 induces Mdm2 transcription. (A, B) RT-PCR analysis of Mdm2 expressions in PC12 cells (A) and cultured cortical neurons (B) after overexpression of SCYL1BP1, BP1-N, or BP1-T. (C) SCYL1-BP1 did not change the half-life of Mdm2. PC12 cells were transfected with or without SCYL1-BP1. At 48 h posttransfection, the cells were treated with 50 μg/ml CHX for the indicated times. Lysates were prepared and analyzed by Western blotting for Mdm2 and the GAPDH loading control. (D) Schematic diagrams showing human and rat genomic DNA structures of Mdm2 with two distinct promoters designated P1 and P2. In human, P1 is a constitutive promoter, and P2 is a p53-responsive promoter that is located between exons 1 and 2 of the gene. An identical length is designated for rat. (E) Dual luciferase assay was performed as described in Materials and Methods . PC12 cells were transfected with a Mdm2 luciferase reporter derived from P1P2, P1, or P2 and plasmids expressing GFP, SCYL1-BP1, BP1-N, or BP1-T, as indicated, and luciferase activity was assayed. The results were normalized to Renilla luciferase activity and represent the mean ± SD of three independent experiments. (F, G) ChIP assays were performed in PC12 cells (F) and cultured cortical neurons (G) as described in Materials and Methods . Rabbit anti–SCYL1-BP1 antibody was used to immunoprecipitate SCYL1-BP1–DNA complexes. Rabbit immunoglobulin G was used as a control.

Article Snippet: To generate the luciferase reporters P1 and P2, a Sac I and Xho I fragment obtained from the P1P2 was cloned into pGL3-Basic (Promega, Madison, WI).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Over Expression, Transfection, Western Blot, Control, Luciferase, Derivative Assay, Expressing, Activity Assay