phosphorylated stat1  (R&D Systems)

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Macrophages were pretreated with GSKJ1 for 30 min, stimulated with IFN-γ for 12 h and subsequently incubated with oxLDL for 24 h. Lipid uptake was evaluated via Oil Red O staining. Scale bars, 200 μm (top) and 100 μm (bottom). b , Macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ for 12 h. Mitochondrial ROS production was detected via a ROS assay kit. Green fluorescence indicates the intensity of the ROS. Scale bar, 100 μm. The macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ for 12 h, and total RNA was subsequently collected for RNA-seq. c , Cnet plot showing KEGG enrichment pathways identified from DEGs between the IFN-γ-treated group and the GSKJ1 + IFN-γ-treated group. d , Heatmap showing DEGs among the control, IFN-γ and GSKJ1 + IFN-γ groups. e , Heatmap showing the enriched metabolic pathways affected by GSKJ1 treatment in macrophages, with differentially regulated pathways marked in red. f , Left: western blot analysis of Stat1, p-Stat1 and Kdm6b in macrophages pretreated with GSKJ1 for 30 min before they were stimulated with IFN-γ at the indicated times, with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Incubation, Staining, ROS Assay, Fluorescence, RNA Sequencing, Control, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ at the indicated times. Left: western blot analysis of Stat1, p-Stat1 and Kdm6b was performed, with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05. b , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Co-IP assessment of the interaction between Stat1 and Kdm6b for the indicated times using an anti-Stat1 antibody. c , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Left: western blot detection of Jak1, phosphorylated Jak1 (p-Jak1) and Kdm6b with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05. d , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Left: co-IP analysis of the interaction between Jak1 and Kdm6b using an anti-Jak1 antibody. Right: the quantified results. * P < 0.05. e , Macrophages were stimulated with IFN-γ for 30 min. IF analysis of Jak1 and Kdm6b in macrophages was performed. Nuclei are visualized with DAPI. Scale bars, 50 μm (left) and 10 μm (right). f , Macrophages were stimulated with IFN-γ for 30 min with or without GSKJ1. Left: co-IP detection of the interaction between Jak1 and Kdm6b using an anti-Jak1 antibody. Right: the quantified results. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Western Blot, Control, Co-Immunoprecipitation Assay

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Heatmap showing the DEGs among differentially abundant neighborhoods in T subsets isolated from atherosclerotic plaques in mice. b , Heatmap showing the DEGs in immune cell subsets isolated from atherosclerotic plaques in mice. c , Chord plot depicting the interactions among immune cell subsets as analyzed by the ComPath method. d , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry analysis of the activated CTL ratio (PD-1 + GZMB + subset). * P < 0.05. e , Macrophages were pretreated with Flu (a STAT1 phosphorylation inhibitor) for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry measurement of the activated CTL ratio. * P < 0.05. f , Macrophages were pretreated with both Flu and GSKJ1 for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry measurement of the activated CTL ratio. * P < 0.05. g , Macrophages were pretreated with IFN-γ for 6 h, followed by αCD80 and GSKJ1 treatment for 6 h before being cocultured with CTLs. Flow cytometry detection of the activated CTL ratio. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Isolation, Flow Cytometry, Phospho-proteomics