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