Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) KEGG enrichment analysis of down-regulated DEGs in Sik2 F/F ; LysM Cre cardiac macrophages on day 3 post-MI. The top 20 enriched pathways are listed. ( B ) DEGs (fold change ≥ 2 and P .adjust ≤ 0.05) overlapped in top 20 of enriched pathways. ( C ) Transcription factor prediction was performed using TRRUST databases. The top 10 transcription factors are listed. ( D ) Immunoblotting analysis and quantification of P65 in the cytoplasm and nucleus of WT and SIK2-knockout PMs and treated with TNF-α (20 ng/ml) or vehicle ( n = 4) for 1 hour. ( E ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression in WT and SIK2-knockout PMs stimulated with TNF-α for 24 hours ( n = 6). ( F ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression ( n = 6). PMs were pretreated with ARN3236 (3 μmol) and stimulated with TNF-α for 24 hours. ( G ) WikiPathways enrichment analysis of differentially phosphorylated proteins in PMs treated with ARN3236 (3 μmol) or control. The top 20 enriched pathways are listed. ( H ) Immunoblotting analysis and quantification of phosphorylated P65 (p-P65), P65, phosphorylated P38 (p-P38), and P38 expression in WT and SIK2-knockout PMs stimulated with TNF-α or vehicle ( n = 6). ( I and J ) Flow cytometry analysis of efferocytosis in WT or LeSik2 Raw264.7 cells [stimulated with SB 203580 (10 μmol) or not] treated with CFSE-prelabeled RBCs ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Analysis, Western Blot, Knock-Out, Expressing, Control, Flow Cytometry, Significance Assay

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Venn diagram delineating the intersection of SIK2-binding protein candidates in the top enriched pathways identified in , that is, NF-κB signaling pathway and MAPK signaling pathway. LC-MS/MS, liquid chromatography–tandem mass spectrometry. ( B ) Top proteins interacting with SIK2 identified through mass spectrometry analysis of peptides pulled down using anti-Flag antibody in HEK293T cells transfected with Flag-Sik2. ( C ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 using anti-Flag antibody, followed by immunoblotting with corresponding antibodies. HA, hemagglutinin. ( D and E ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 and HA-Traf2 using anti-Flag (D) or anti-HA (E) antibody, followed by immunoblotting with corresponding antibodies. FL, full length. ( F ) Immunoprecipitation (IP) in HEK293T cells cotransfected with HA-Traf2 and truncated Flag-Sik2 using an anti-HA antibody, followed by immunoblotting (IB) with corresponding antibodies. ( G ) Immunoprecipitation in HEK293T cells cotransfected with Flag-Sik2 and truncated HA-Traf2 using an anti-HA antibody, followed by immunoblotting with corresponding antibodies. ( H ) Western blotting analysis and quantification of SIK2, TRAF2, p-P38, P38, p-P65, and P65 expression in HEK293T cells transfected with SIK2 and si-TRAF2 or control ( n = 4). ( I ) Coimmunoprecipitation (Co-IP) analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and His-Ub, along with Flag-Sik2 or Flag-Sik2 T175A mutant. ( J ) Co-IP analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and Flag-Sik2, along with Myc-K48-Ub or Myc-K63-Ub. ( K ) Co-IP analysis of TRAF2 phosphate serine (p-Ser) and phosphate threonine (p-Thr) in HEK293T cells transfected with HA-Traf2, along with Flag-Sik2 or Flag-Sik2 T175A mutant.

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Analysis, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Mutagenesis, Tandem Mass Spectroscopy

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) KEGG enrichment analysis of down-regulated DEGs in Sik2 F/F ; LysM Cre cardiac macrophages on day 3 post-MI. The top 20 enriched pathways are listed. ( B ) DEGs (fold change ≥ 2 and P .adjust ≤ 0.05) overlapped in top 20 of enriched pathways. ( C ) Transcription factor prediction was performed using TRRUST databases. The top 10 transcription factors are listed. ( D ) Immunoblotting analysis and quantification of P65 in the cytoplasm and nucleus of WT and SIK2-knockout PMs and treated with TNF-α (20 ng/ml) or vehicle ( n = 4) for 1 hour. ( E ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression in WT and SIK2-knockout PMs stimulated with TNF-α for 24 hours ( n = 6). ( F ) qPCR analysis of Il-1β , Il-6 , Nos2 , Cxcl-1 , and Mmp-9 mRNA expression ( n = 6). PMs were pretreated with ARN3236 (3 μmol) and stimulated with TNF-α for 24 hours. ( G ) WikiPathways enrichment analysis of differentially phosphorylated proteins in PMs treated with ARN3236 (3 μmol) or control. The top 20 enriched pathways are listed. ( H ) Immunoblotting analysis and quantification of phosphorylated P65 (p-P65), P65, phosphorylated P38 (p-P38), and P38 expression in WT and SIK2-knockout PMs stimulated with TNF-α or vehicle ( n = 6). ( I and J ) Flow cytometry analysis of efferocytosis in WT or LeSik2 Raw264.7 cells [stimulated with SB 203580 (10 μmol) or not] treated with CFSE-prelabeled RBCs ( n = 6).

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Analysis, Western Blot, Knock-Out, Expressing, Control, Flow Cytometry, Significance Assay

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Venn diagram delineating the intersection of SIK2-binding protein candidates in the top enriched pathways identified in , that is, NF-κB signaling pathway and MAPK signaling pathway. LC-MS/MS, liquid chromatography–tandem mass spectrometry. ( B ) Top proteins interacting with SIK2 identified through mass spectrometry analysis of peptides pulled down using anti-Flag antibody in HEK293T cells transfected with Flag-Sik2. ( C ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 using anti-Flag antibody, followed by immunoblotting with corresponding antibodies. HA, hemagglutinin. ( D and E ) Immunoprecipitation in HEK293T cells transfected with Flag-Sik2 and HA-Traf2 using anti-Flag (D) or anti-HA (E) antibody, followed by immunoblotting with corresponding antibodies. FL, full length. ( F ) Immunoprecipitation (IP) in HEK293T cells cotransfected with HA-Traf2 and truncated Flag-Sik2 using an anti-HA antibody, followed by immunoblotting (IB) with corresponding antibodies. ( G ) Immunoprecipitation in HEK293T cells cotransfected with Flag-Sik2 and truncated HA-Traf2 using an anti-HA antibody, followed by immunoblotting with corresponding antibodies. ( H ) Western blotting analysis and quantification of SIK2, TRAF2, p-P38, P38, p-P65, and P65 expression in HEK293T cells transfected with SIK2 and si-TRAF2 or control ( n = 4). ( I ) Coimmunoprecipitation (Co-IP) analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and His-Ub, along with Flag-Sik2 or Flag-Sik2 T175A mutant. ( J ) Co-IP analysis of TRAF2 ubiquitination in HEK293T cells cotransfected with HA-Traf2 and Flag-Sik2, along with Myc-K48-Ub or Myc-K63-Ub. ( K ) Co-IP analysis of TRAF2 phosphate serine (p-Ser) and phosphate threonine (p-Thr) in HEK293T cells transfected with HA-Traf2, along with Flag-Sik2 or Flag-Sik2 T175A mutant.

Article Snippet: Primary antibodies used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Analysis, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Mutagenesis, Tandem Mass Spectroscopy

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet:

Article Snippet: Anti-p65 rabbit antibody , Proteintech , 10745-1-AP; RRID: AB_2178878.

Techniques: Cell-Signaling, Virus, Tissue, Microarray, Recombinant, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software, Biomarker Discovery, In Vivo Imaging