p44 ddk  (OriGene)

Journal: Journal of neurochemistry

Article Title: The endoplasmic reticulum acetyltransferases ATase1/NAT8B and ATase2/NAT8 are differentially regulated to adjust engagement of the secretory pathway

doi: 10.1111/jnc.14958

Figure Lengend Snippet: ATase1 and ATase2 exhibit additional modes of transcriptional regulated. (A) Schematic view of predicted, in silico p53 TFBSs of human ATase1 and ATase2. Predicted TFBS locations are as follows: ATase1: −242 to −223, −451 to −426, −630 to −611, −965 to −946; ATase2: −179 to −173, −219 to −200, −594 to −575. The italicized labels represent 200 bp portions of the promoter that can be amplified by flanking primers. (B) HEK293 cell chromatin after mechanical shearing used for ChIP analysis. (C) ChIP analysis of HEK293 cell chromatin using an antibody targeting p53. The PCR was performed on chromatin isolated before (input) and after immunoprecipitation using either the target antibody or rabbit IgG (negative control). The primer sets used are those that produce the amplicons designated in A. (D) Western blotting of H4 cell nuclear protein before (input) and after pulldown by double stranded DNA probes consisting of the promoter fragments designated in A. (E) Western blotting of Saos2 cell lysate after transfection with DDK-tagged p44 or p53. (F-G) Dual luciferase reporter assay in Saos2 cells as described before. The data are represented as mean + SD, n = 3 independent cell culture preparations. (H) RT-qPCR comparing the relative mRNA expression of ATase1 in Saos2 cells transiently transfected with empty vector (control), p44, or p53. The data are represented as mean + SD, n = 3 independent cell culture preparations. Number of technical replicate outliers removed: p44 transfection probing for ATase1 (1). (I) ChIP analysis of H4 cell chromatin using an antibody targeting REST. The PCR was performed on chromatin isolated before (input) and after immunoprecipitation using either the target antibody or rabbit IgG (negative control). (J) Western blotting of H4 cell nuclear protein before (input) and after pulldown by double stranded DNA probes consisting of the promoter fragments in A. Snap25 was used as a positive control. (K) RT-qPCR comparing the relative mRNA expression of AT-1, ATase1, and ATase1 in cells transiently transfected with empty vector (control) or REST. The data are represented as mean + SD, n = 3 independent cell culture preparations. Number of technical replicate outliers removed: REST transfection probing for ATase1 (1). *p < 0.05, **p < 0.005, ***p < 0.0005 (vs. control).

Article Snippet: Mammalian Cell Transfection H4, PC12, or Saos2 cells were transiently transfected with Lipofectamine 2000 (Invitrogen #11668019; 2017) using the following constructs: human CREB1-pCMV6 (Origene #RC210577; 2017), human c-FOS-pCMV6 (Origene #RC202597; 2017), human c-JUN-pCMV6 (Origene #RC209804; 2017), p53-pCMV6 (OriGene #RC200003; 2019), p44-DDK ( Pehar et al. 2014 ), LPC-flag-REST (RRID:Addgene_41903), LPC-flag empty vector (made in-house via BamHI restriction and re-ligation), or pCMV6-entry empty plasmid (OriGene #PS100001; 2017); cells were harvested 48 hours after transfection.

Techniques: In Silico, Amplification, Isolation, Immunoprecipitation, Negative Control, Western Blot, Transfection, Luciferase, Reporter Assay, Cell Culture, Quantitative RT-PCR, Expressing, Plasmid Preparation, Positive Control