rabbit against mono methyl arginine  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: The arginine methyltransferase PRMT7 promotes extravasation of monocytes resulting in tissue injury in COPD

doi: 10.1038/s41467-022-28809-4

Figure Lengend Snippet: a – d Mono-methylated proteins were immunoprecipitated by anti-mono-methylarginine antibody from whole-cell lysates of Prmt7 +/+ and Prmt7 null MH-S macrophage cells and analyzed by LC-MS/MS proteomics ( n = 3 pull-downs per cell line). a Schematic representation of the experiment. b mRNA expression level of Prmt7 determined by qPCR in Prmt7 +/+ and Prmt7 null MH-S macrophage cells generated by CRISPR/Cas9-mediated targeting of Prmt7 ( n = 9 replicates per cell line, ND not detectable). c Western blot analysis of PRMT7 expression in Prmt7 +/+ and Prmt7 null MH-S cells (repeated two times). d Immunoprecipitation intensity of core histones H2B, H3, H4, and linker H1.2 and H1.5 (Individual pull-downs shown). e Top enriched KEGG pathways under the immune system, cell community, and signal transduction following InCroMAP analysis of the differentially pulled down proteins with less abundance in Prmt7 null compared to Prmt7 +/+ MH-S cells (FC <−1.6). P values were calculated using a hypergeometric test embedded in the InCroMAP software, no testing for multiple correction. f The fold change of trans-well migrating Prmt7 +/+ and Prmt7 null MH-S macrophage cells in 24 h towards serum-free (SF) medium ± 100 ng/ml CCL2 ( n = 4, repeated two times). g Wound migration assay in Prmt7 +/+ ( n = 7) and Prmt7 null ( n = 6) MH-S cells grown to confluence and scratched. Representative images at 0 and 24 h post scratch plus the percentage wound closure at 24 h (repeated twice). Scale bar, 200 μm. h WST assay of Prmt7 +/+ ( n = 3) and Prmt7 null ( n = 3) MH-S cells at 24 h. i Percentage of attached Prmt7 +/+ and Prmt7 null MH-S cells at the time points indicated post-seeding ( n = 2 per cell line, repeated twice). j mRNA expression levels of ITGAL and ITGAM determined by qPCR in the monocytes of smokers ( n = 11) and non-smokers ( n = 10) isolated from the peripheral blood. k MFI of ITGAL and ITGAM surface expression as determined by flow cytometry in Prmt7 +/+ ( n = 3) and Prmt7 null ( n = 4) MH-S cells. l MFI of ITGAL and ITGAM surface expression as determined by flow cytometry in WT MH-S cells incubated with 5 μM SGC3027 (PRMT7 inhibitor) for 24 h and analyzed ( n = 2). m Western blot analysis of phosphorylated ERK and p38 in Prmt7 +/+ and Prmt7 null MH-S cells incubated for 30 min with 100 ng/ml CCL2 (repeated independently two times). n Western blot analysis of phosphorylated ERK and p38 in Prmt7 +/+ and Prmt7 null MH-S cells incubated for 15 min with 1 μg/ml LPS (repeated independently two times). Data shown mean ± SD, P values shown in charts determined by one-way ANOVA Bonferroni’s multiple comparisons test ( f , i ), unpaired two-tailed Student’s t -test ( b , d , g , j – l ). Source data are provided as a Source Data file.

Article Snippet: After blocking, cells were incubated with primary antibodies against mono-methylated arginine (Cat. No. 8015, 1:100, Cell Signaling Technology), Total H3 (Cat. No. ab195277, 1:100, Abcam) and RAP1A/B (Cat. No. ab175329, 1:50, Abcam) for 1 h at 37 °C.

Techniques: Methylation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Generated, CRISPR, Western Blot, Transduction, Software, Migration, WST Assay, Isolation, Flow Cytometry, Incubation, Two Tailed Test