Journal: eLife
Article Title: Gαq-PKD/PKCμ signal regulating the nuclear export of HDAC5 to induce the IκB expression and limit the NF-κB-mediated inflammatory response essential for early pregnancy
doi: 10.7554/eLife.83083
Figure Lengend Snippet: ( A ) Western blot analysis of the p65 and p50 expression in cytoplasm and nucleus of CON and KO human endometrial stromal cell (HESC) treated with differential culture medium for 4 days, nuclear translocation of p65 and p50 were higher in GNAQ-KO decidual cells than in control. GAPDH and histone 3 (H3) were used as the loading controls. ( B ) Immunofluorescence staining of p65 in CON and KO stromal cells and treated with differential culture medium for 4 days, nuclear translocation p65 were higher in GNAQ-KO decidual cells than in control. Scale bars, 100 μm. ( C ) Western blot analysis of p-p65 expression in whole-cell lysates of CON and KO cells treated with differential culture medium for 2, 4, and 6 days, phosphorylation of the nuclear factor (NF)-κB subunit p65 were higher in GNAQ-KO decidual cells than in control. GAPDH was used as the loading controls. ( D, E ) qRT-PCR analysis of IL-11 and IL-1β mRNA expression levels in CON and KO HESC treated as in ( C ) ( n = 5). ( F ) Western blot analysis of p-STAT3, STAT3, and IL-1β expression in whole-cell lysates of CON and KO HESC treated as in ( C ). β-Tubulin was used as the loading control. ( G, H, J, K ) qRT-PCR analysis of IL-11 , IL-1β , PRL , and IGFBP-1 mRNA after treatment with NF-κB inhibitor, pyrrolidinedithiocarbamate ammonium (PDTC, 10 μM), in CON and KO HESC treated with differential culture medium for 4 days ( n = 3). ( I, L ) Western blot analysis of p-STAT3, IL-1β, and IGFBP-1 expression in whole-cell lysates of differentiated CON and KO HESC treated as in ( G ). GAPDH was used as the loading control. Representative data are shown from three to five independent experiments. ( D and E ) were analyzed with unpaired Student’s t -test. *p < 0.05 . ( G, H, J, and K ) were calculated with one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests. *p < 0.05. Figure 2—source data 1. Original blot images of .
Article Snippet: The membrane was blocked in 5% skimmed milk (BD) and probed with rabbit polyclonal antibody against Gαq, rabbit monoclonal antibody against IGFBP-1 (Abcam, Cat# ab181141, 1:1000), mouse monoclonal antibody against IL-1β (Origene Inc, Cat# TA506443, 1:1000), rabbit polyclonal antibody against p65 (Abcam, Cat# ab32536, 1:1000), rabbit polyclonal antibody against p50 (Abcam Cat# ab32360, 1:1000), p-STAT3 (Abcam Cat# ab76315, 1:1000), STAT3 (Abcam Cat# ab68153, 1:1000), rabbit polyclonal antibody against IκBa (Abcam Cat# ab32518, 1:1000), rabbit polyclonal antibody against p-NFκB p65(Ser536) (Bioss, Cat# bs-0982R, 1:1000), mouse monoclonal antibody against p-IκBа (Ser32) (Bioss, Cat# bsm-52169R, 1:1000), rabbit monoclonal antibody against p-PKA C(Thr197) (Cell Signaling Technology, Cat# 5661, 1:1000), rabbit monoclonal antibody against PKA C-а (Cell Signaling Technology, Cat# 5842, 1:1000), rabbit polyclonal antibody against p-PKCа/βǁ (Thr638/641) (Cell Signaling Technology, Cat# 9375, 1:1000), rabbit polyclonal antibody against PKCа (Cell Signaling Technology, Cat# 2056, 1:1000) and p-PKC Antibody Sampler Kit (Cell Signaling Technology, Cat# 9921, 1:1000), mouse monoclonal antibody against HDAC5 (Cell Signaling Technology, Cat# 98329, 1:1000), and rabbit polyclonal antibody against p-HDAC5 (ABclonal, Cat# AP0202, 1:1000).
Techniques: Western Blot, Expressing, Translocation Assay, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Comparison