Fig 1: PADI4 mediates expression of the Elk-1 target gene c-Fos.(A, C and D) Real-time RT-PCR analysis of the c-Fos and GAPDH expression in serum-starved or EGF-stimulated MCF-7 cells (A) with or without Cl-Amidine or U0126 treatment; (C) with or without PADI4 knockdown; (D) with or without Elk-1 knockdown. Expression data are normalized to ß-actin transcripts and the graph represents the mean + SEM (n = 3). *P<0.05, **P<0.01. (B) Dose-dependent effect of Cl-Amidine treatment on Elk-1 activation and ERK2 phosphorylation in MCF-7 cells. Different doses of Cl-Amidine were added to the normal MCF-7 cell culture media for 48 hours and western blots of cell lysates were then performed using anti-p-Elk-1, anti-Elk-1, anti-p-ERK and ERK2 antibodies. The graph on the right is a semi-quantitative analysis of the western blots (relative intensity) with an arbitrary number of the intensity ratio of p-Elk-1 compared to total Elk-1, or p-ERK compared to total ERK2 using Image J software. (E) Stable overexpression (OE) of PADI4 in MCF-7 cells increases transcriptional output of c-Fos. Left: qRT-PCR analysis showing that PADI4 expression is increased in PADI4 OE MCF-7 cells. Right: qRT-PCR analysis showing that c-Fos transcription output was significantly elevated in PADI4 OE MCF-7 cells under either control or EGF treatment. (F) PADI4 enzymatic activity facilitates Elk-1 mediated gene transcription. Expression vector for the DNA-binding domain of GAL4 (CMV-GAL4) or GAL4-Elk-1 was transfected into HEK293 cells along with the 5xGAL4-driven luciferase reporter plasmid containing the minimal E1b promoter and plasmids for pcDNA3.1, wild type (WT) or inactive mutant (C645S) PADI4 with N-terminal Flag tags. Luciferase activity was determined per microgram total cell protein 24 hours after transfection and results are represented as fold relative to empty vector control. Data shown are representative of two independent experiments performed in triplicate and error bars indicate SEM. *P<0.05 (two-tailed paired Student's t-test). In the lower panel, the expression of Flag-PADI4 and GAL4-Elk-1 fusion proteins was monitored by western blot using anti-Flag and anti-GAL4 antibodies.
Fig 2: PADI4 is enriched in the gene promoter region near the TSSs.(A) Scatterplot showing the mean log2 ratio of each window on the array for two PADI4 ChIP-chip replicates. The spearman rank correlation coefficient and associated p-value are indicated. (B) Heat map of PADI4 ChIP-chip data for 19,206 promoters from -2,200 bp to +500 bp relative to the TSS. The values in the x-axis represent the center of a 600 bp window with 150 bp steps relative to each TSS. The promoters are ordered top to bottom based on the increasing intensity of the PADI4 signal at the promoter surrounding the TSSs. (C) Histogram showing the number of statistically significant peaks (Wilcoxon signed-rank test, p<0.016) across the entire tiled region for all the promoters on the ChIP-chip array. The asterisk denotes significant enrichment of PADI4 peaks near the TSSs based on the p-value from a Fisher exact test (*p = 0.0444, **p = 0.0085, ***p = 6.21×10-9, ****p = 1.42×10-15).
Fig 3: PAD2 expression in cultured RAW 264.7 macrophage cells. (A) RT-PCR for PAD2 and PAD4 mRNA expression in cultured RAW 264.7 macrophages. Two unique primer sequence sets were used to test for PAD2 and PAD4 mRNA expression levels. RAW 264.7 macrophages were found to strongly express PAD2 mRNA. GAPDH mRNA expression levels were used as the internal control for RT-PCR. (B) Anti-PAD2 immunofluorescence labeling shows positive staining in the nucleus and cytoplasm of RAW 264.7 macrophages. Nuclei are stained with DAPI.
Fig 4: Expression of PAD2 and PAD4 in non-malignant and tumoral canine and feline mammary cell lines. a Expression levels of the genes PAD2 and PAD4 in canine and feline normal and tumoral mammary cell lines as determined by qRT-PCR. b Protein expression in whole cell lysates subjected to SDS-PAGE and immunoblot analyses probed with PAD2 or PAD4 antibodies. ß-actin was included as a loading control. Representative Western blots and quantifications are shown. Quantification is represented as the fold change of PAD2/4 band density over ß-actin band density. c Protein expression in cells probed with either PAD2 or PAD4 antibodies and subjected to flow cytometry analyses. d Protein expression in whole cell lysates subjected to SDS-PAGE and immunoblot analyses probed with anti-modified citrulline antibodies. ß-actin was included as a loading control. Representative Western blots and quantifications are shown. Quantification is represented as the fold change of modified-citrulline band density over ß-actin band density. *P < 0.05, n = 3. Data are presented as mean ± standard deviation
Fig 5: Working model depicting role of PADI4 in Elk-1–mediated activation of c-Fos.(A) Prior to stimulation, Elk-1, PADI4, and p300 constitutively occupy the c-Fos promoter. (B) Following EGF treatment, Elk-1 is citrullinated by PADI4. (C) Upon citrullination, Elk-1 is then phosphorylated by ERK2. (D) This phosphorylation event leads to an enhanced interaction between Elk-1 and p300 (9). (E) The enhanced interaction then potentiates p300-mediated histone acetylation and subsequent transcriptional activation.
Supplier Page from MilliporeSigma for Anti-PADI4 (N-terminal) antibody produced in rabbit