phospho cjun ser63  (Santa Cruz Biotechnology)

Journal: Cell Death Discovery

Article Title: Atorvastatin improves spermatogenesis in murine and in vitro human chronic orchitis models through restoring blood-testis barriers

doi: 10.1038/s41420-025-02749-6

Figure Lengend Snippet: a Venn diagram of differential expressed proteins (DEPs) between groups. b Enriched GO terms of the co-downregulated proteins in Vehicle and T + AT groups compared with TNF-α group. c Heatmap showing the top 10 co-downregulated proteins according to the fold change between T + AT group and TNF-α group. d Western blot images of MMP3 and MMP9 in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups. e Grayscale value analysis of western blot for MMP3 and MMP9 in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups, n = 3. f , g Immunofluorescence of MMP3 and MMP9 (green) co-stained with SOX9 (red) in Vehicle, EAO-Sal and EAO-AT groups and quantitative analysis for immunofluorescence intensity of MMP3 and MMP9 in SOX9 + Sertoli cells from 20 randomly selected fields. SOX9 labeled Sertoli cells. Scale bar, 10 μm. n = 4 testis samples per group. h Western blot assay of Rac1, cJUN and Phospho-cJUN (p-cJUN) in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups. i Grayscale value analysis of western blot for Rac1 and p-cJUN in TM4 Sertoli cells from Vehicle, TNF-α and T + AT groups, n = 3. j Immunofluorescence of cJUN (green) co-stained with SOX9 (red) in Vehicle, EAO-Sal and EAO-AT groups and quantitative analysis for immunofluorescence intensity of cJUN in the nucleus of SOX9 + Sertoli cells from 20 randomly selected fields. SOX9 labeled Sertoli cells. Scale bar, 10 μm. k Western blot images of MMP3, MMP9, CX43 and ZO1 in TM4 Sertoli cells after T-5224 treatment. Data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, **** P < 0.0001, ns indicates no statistical significance.

Article Snippet: The primary antibodies used in this study were listed: rabbit anti-HMGCR (A19063, ABclonal, 1:1000), rabbit anti-CX43 (26980-1-AP, Proteintech, 1:1000), rabbit anti-ZO1 (21773-1-AP, Proteintech, 1:1000), rabbit anti-CTNNB1 (ab32572, Abcam, 1:1000), rabbit anti-MMP3 (17873-1-AP, Proteintech, 1:1200), rabbit anti-MMP9 (10375-2-AP, Proteintech, 1:1200), rabbit anti-cJUN (ab32137, Abcam, 1:4000), rabbit anti-p cJUN (9261S, Cell Signaling Technology, 1:1000), rabbit anti-Rac1 (24072-1-AP, Proteintech, 1:1000).

Techniques: Western Blot, Immunofluorescence, Staining, Labeling

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) The lack of Myc mRNA downregulation after prolonged TNF stimulation in B6.Sst1S macrophages. BMDMs from B6 and B6.Sst1S were treated with 10 ng/mL TNF for 6, 12, and 24 hr. Expression of Myc was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated B6 BMDMs. β-actin was used as the internal control. ( B ) Myc protein levels expressed by B6 and B6.Sst1S BMDMs during the course of stimulation with TNF(10 ng/mL) for 6 and 12 hr. (western blot). Average densitometric values from two independent experiments were included above the blot. ( C ) Myc inhibition restored the levels of Fth and Ftl proteins in TNF-stimulated B6.Sst1S macrophages to the B6 levels. B6 and B6.Sst1S BMDMs were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Protein levels of Fth and Ftl were observed using western blot. Average densitometric values from two independent experiments were included above the blot. ( D ) Myc inhibition decreased the labile iron pool in TNF-stimulated B6.Sst1S macrophages. B6.Sst1S BMDMs were treated with 10 ng/mL TNF or left untreated for 48 hr. The 10058-F4 inhibitor was added 2 hr post TNF stimulation. The labile iron pool (LIP) was measured using the Calcein AM method and represented as fold change as compared to untreated control. DFO was used as a negative control, and FeSO4 was used as a positive control. ( E and F ) Myc inhibition reduced lipid peroxidation in TNF-stimulated B6.Sst1S BMDMs. Cells were treated with 10 ng/mL TNF in the presence or absence of 10058-F4 for 48 hr. The inhibitor was added 2 hr post TNF stimulation. The MDA production was measured using commercial MDA assay ( E ) The lipid peroxidation was measured by fluorometric method using C11-Bodipy 581/591 ( F ). ( G ) B6.Sst1S BMDMs were treated as above in E. The accumulation of lipid peroxidation product, 4-HNE after 48 hr was detected by confocal microscopy using 4-HNE-specific antibody. The 4-HNE adducts accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated group. ( H ) The BMDMs from B6.Sst1S were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Expression of Ifnb1 , Rsad2, Trib3, and Chac1 was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated group. 18 S was used as the internal control. ( I and J ) B6 ( I ) and B6.Sst1S ( J ) BMDMs were treated with TNF (10 ng/ml) for 6, 12, and 24 hr in the presence or absence of JNK inhibitor D-JNK1 (2 μM). The cells were harvested and the protein levels of c-Myc and p-cJun were determined by western blotting. JNK inhibitor D-JNK1 was added 2 hr post TNF stimulation. Average densitometric values from two independent experiments were included above the blot. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by two-way ANOVA using Šídák’s multiple comparison test (Panel A) and ordinary one-way ANOVA using Šídák’s multiple comparison test (Panels D-F and H). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 5—source data 1. PDF file containing original western blots for indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Rabbit polyclonal anti-phospho-cJun antibody , Cell Signaling Technology , Cat# 9261 s, RRID: AB_2130162 , WB (1:1000).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Inhibition, Negative Control, Positive Control, Multiple Displacement Amplification, Confocal Microscopy, Standard Deviation, Comparison