Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a WCLs derived from HSC-3 cells were immunoprecipitated with control IgG, anti-PA28γ, or anti-CK2β antibody, followed by IB analysis of co-IP products and WCLs. b Immunofluorescence analyses of the cellular localization of endogenous PA28γ and CK2β in HNSCC cells. DAPI (blue), nucleus; Scale bars, 10 μm. c Schematic representation of the evolutionarily conserved PA28γ-T23 site in different species. Residues around the T23 site have a common characteristic of CK2 kinase substrates, namely the predominance of acidic amino acid residues (E/D) between n-4 and n + 7. α1–4 denotes helix-α1–4. d IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids. e In vitro kinase assays were performed with recombinant His-PA28γ WT and T23A proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. IB analysis of the indicated proteins in vitro kinase assay samples. f, g IB analysis of WCLs derived from HEK293T cells transfected with the subunits of CK2 ( f ) and HNSCC cells treated with 100ngml –1 EGF before harvesting ( g ). h, i HNSCC cells silenced with siRNA negative control (siNC) or siCK2 ( h ) and HSC-3 cells silenced with siNC or CK2α/α‘/β siRNA ( i ) were stimulated without or with 100ngml –1 EGF for 30 min before harvesting for IB analysis. j HEK293T cells transfected with the indicated plasmids were treated with 100ngml –1 EGF. WCLs were immunoprecipitated with the indicated antibodies, followed by IB analysis of co-IP products and WCLs. k In vitro kinase assays were performed with recombinant His-PA28γ proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. 2 μM or 20 μM CK2 inhibitor TBB was added as indicated. l IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. m IB analysis of WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. Source data are provided as a Source Data file.

Article Snippet: Anti-His (IB, 1:10000; IP, 1:300; 66005-1-Ig), anti-GST (IB, 1:10000; 66001-2-Ig), anti-NIP30 (IB, 1:1000; 16830-1-AP), anti-SRC3 (IB, 1:3000; 29587-1-AP), anti-SirT1 (IB, 1:3000; 13161-1-AP), anti-pan-keratin (pan-K) (IHC, 1:3000; 26411-1-AP), anti-Ki67 (IHC, 1:5000; 27309-1-AP) and anti-CK2β (IHC, 1:200; 20234-1-AP) antibodies were purchased from Proteintech.

Techniques: Derivative Assay, Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, In Vitro, Recombinant, Purification, Kinase Assay, Negative Control

Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a WCLs derived from HSC-3 cells were immunoprecipitated with control IgG, anti-PA28γ, or anti-CK2β antibody, followed by IB analysis of co-IP products and WCLs. b Immunofluorescence analyses of the cellular localization of endogenous PA28γ and CK2β in HNSCC cells. DAPI (blue), nucleus; Scale bars, 10 μm. c Schematic representation of the evolutionarily conserved PA28γ-T23 site in different species. Residues around the T23 site have a common characteristic of CK2 kinase substrates, namely the predominance of acidic amino acid residues (E/D) between n-4 and n + 7. α1–4 denotes helix-α1–4. d IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids. e In vitro kinase assays were performed with recombinant His-PA28γ WT and T23A proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. IB analysis of the indicated proteins in vitro kinase assay samples. f, g IB analysis of WCLs derived from HEK293T cells transfected with the subunits of CK2 ( f ) and HNSCC cells treated with 100ngml –1 EGF before harvesting ( g ). h, i HNSCC cells silenced with siRNA negative control (siNC) or siCK2 ( h ) and HSC-3 cells silenced with siNC or CK2α/α‘/β siRNA ( i ) were stimulated without or with 100ngml –1 EGF for 30 min before harvesting for IB analysis. j HEK293T cells transfected with the indicated plasmids were treated with 100ngml –1 EGF. WCLs were immunoprecipitated with the indicated antibodies, followed by IB analysis of co-IP products and WCLs. k In vitro kinase assays were performed with recombinant His-PA28γ proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. 2 μM or 20 μM CK2 inhibitor TBB was added as indicated. l IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. m IB analysis of WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. Source data are provided as a Source Data file.

Article Snippet: Anti-CK2β (IB, 1:300; IP, 1:50; IF, 1:50; sc-12739), anti-CK2α (IB, 1:200; sc-12738), anti-CK2α‘ (IB, 1:200; sc-514403), anti-HA (IB, 1:200; sc-7392) and anti-E4F1 (IB, 1:100; IP, 1:50; sc-514718) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, In Vitro, Recombinant, Purification, Kinase Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Macrophage Tim-4 protects against deep vein thrombosis by binding CK2β to suppress inflammatory responses

doi: 10.3389/fimmu.2025.1634230

Figure Lengend Snippet: Tim-4 hijacks CK2β to prevent the activation of the NF-κB pathway. (A) Tim-4 binding proteins in PEMs were identified by RNA-Seq combined with LC-MS/MS assay. (B) Predicted docking module of Tim-4 and CK2β. (C, D) IP validation of Tim-4 binding to CK2β in PEMs. (E) Co-IP assay using either anti-HA or anti-Flag antibodies with lysates from HEK293T cells 48 hours after co-transfection with HA-tagged Tim-4 and Flag-tagged CK2β plasmids. (F, G) Representative colocalization IF images of Tim-4 and CK2β in HEK293T cells or PEMs. Scale bar, 10μm. (H-K) THP-1 cells were transfected with Lv-shTim-4 or Lv-NC for 72 hours and treated with LPS for 0.5 hours, and inhibitor groups were treated with CX-4945 for another 1 hour and co-cultured with HUVECs. (H, I) Relative mRNA and protein levels of vWF and TF in HUVECs. (J, K) Relative mRNA levels of IL-1β and TNF-α, and western blot analysis of pP65, P65, pIκBα, and IκBα expression in THP-1. (L-O) Representative ultrasound images and photographs (Scale bar, 1 mm) of thrombi, weight statistics, and HE staining of thrombi (scale bar, 500 μm) and spleens (scale bar, 0.100 mm) from Tim-4 fl/fl and LysM-Cre; Tim-4 fl/fl mice with or without CX-4945 treatment. (P) Western blot analysis of vWF and TF expression in IVCs from Tim-4 fl/fl and LysM-Cre; Tim-4 fl/fl mice with or without CX-4945 treatment. (Q) qPCR analysis for IL-1β and TNF-α in PEMs and BMDMs from Tim-4 fl/fl and LysM-Cre; Tim-4 fl/fl mice with or without CX-4945 treatment. (R) Western blot analysis of pP65, P65, pIκBα, and IκBα expression in PEMs and BMDMs from Tim-4 fl/fl and LysM-Cre; Tim-4 fl/fl mice with or without CX-4945 treatment. Error bars indicate SD of at least three biological replicates per group in one experiment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (A8020, Solarbio, Beijing, China) at room temperature for 2 hours and then incubated with the appropriate primary antibodies against Tim-4 (12008-1-AP, Proteintech, Wuhan, China), CK2β (GB113852-50, Servicebio, Wuhan, China), TF (CY5807, Abways, Shanghai, China), vWF (11778-1-AP, Proteintech, Wuhan, China), P65 (CY5034, Abways, Shanghai, China), pP65 (3033S, Cell Signaling Technology, Danvers, MA, USA), IκBα (10268-1-AP, Proteintech, Wuhan, China), pIκBα (82349-1-RR, Proteintech, Wuhan, China), and GAPDH (AB0037, Abways, Shanghai, China) overnight at 4°C.

Techniques: Activation Assay, Binding Assay, RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery, Co-Immunoprecipitation Assay, Cotransfection, Transfection, Cell Culture, Western Blot, Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Macrophage Tim-4 protects against deep vein thrombosis by binding CK2β to suppress inflammatory responses

doi: 10.3389/fimmu.2025.1634230

Figure Lengend Snippet: Confirmation of Tim-4 and associated molecule expression in clinical specimens. (A-D) qPCR assay for Tim-4, lnc219, miR-93-5p, IL-1β and TNF-α expression in PBMCs from DVT patients compared with control subjects at mRNA level. (E-G) IF staining and quantification of Tim-4 (green) and CD68 (red), CK2β (green) and CD68 (red), CK2β (green) and Tim-4 (red) respectively in popliteal vein tissues with or without thrombosis. Scale bar, 10 μm. Error bars indicate SD of at least three biological replicates per group in one experiment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (A8020, Solarbio, Beijing, China) at room temperature for 2 hours and then incubated with the appropriate primary antibodies against Tim-4 (12008-1-AP, Proteintech, Wuhan, China), CK2β (GB113852-50, Servicebio, Wuhan, China), TF (CY5807, Abways, Shanghai, China), vWF (11778-1-AP, Proteintech, Wuhan, China), P65 (CY5034, Abways, Shanghai, China), pP65 (3033S, Cell Signaling Technology, Danvers, MA, USA), IκBα (10268-1-AP, Proteintech, Wuhan, China), pIκBα (82349-1-RR, Proteintech, Wuhan, China), and GAPDH (AB0037, Abways, Shanghai, China) overnight at 4°C.

Techniques: Expressing, Control, Staining

Journal: Frontiers in Immunology

Article Title: Macrophage Tim-4 protects against deep vein thrombosis by binding CK2β to suppress inflammatory responses

doi: 10.3389/fimmu.2025.1634230

Figure Lengend Snippet: Schematic diagram of lncRNF219-3:1/miR-93-5p regulating Tim-4 in the pathogenesis of DVT by hijacking CK2β. In Tim-4 fl/fl mice, highly expressed Tim-4 can inhibit kinase activity of CK2 holoenzyme by hijacking and interacting with CK2β, and diminish the phosphorylation of P65 and IκBα, thereby inhibiting NF-κB signaling pathway activation-induced DVT progression. In addition, highly expressed lnc219 acting as a competing endogenous RNA, can increase Tim-4 expression by sponging miR-93-5p and enhance Tim-4-mediated anti-inflammatory responses and anti-thrombosis effects. In lysM-Cre; Tim-4 fl/fl mice, Tim-4 deficiency abolishes the interaction with CK2β, leading to activation of CK2 dependent NF-κB signaling pathway, and eventually aggravates inflammatory factors-mediated DVT development.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (BSA) (A8020, Solarbio, Beijing, China) at room temperature for 2 hours and then incubated with the appropriate primary antibodies against Tim-4 (12008-1-AP, Proteintech, Wuhan, China), CK2β (GB113852-50, Servicebio, Wuhan, China), TF (CY5807, Abways, Shanghai, China), vWF (11778-1-AP, Proteintech, Wuhan, China), P65 (CY5034, Abways, Shanghai, China), pP65 (3033S, Cell Signaling Technology, Danvers, MA, USA), IκBα (10268-1-AP, Proteintech, Wuhan, China), pIκBα (82349-1-RR, Proteintech, Wuhan, China), and GAPDH (AB0037, Abways, Shanghai, China) overnight at 4°C.

Techniques: Activity Assay, Phospho-proteomics, Activation Assay, Expressing