anti-ck2β antibody e9  (Santa Cruz Biotechnology)

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: Bioscience Reports

Article Title: Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP 7

doi: 10.1042/BSR20240792

Figure Lengend Snippet: ( A,B ) Effect of CK2 activity on the level of AP3B1. Myc-tagged AP3B1, V5-tagged IP6K1, and active (A) or catalytically inactive Lys68Met (B) HA-tagged CK2α were expressed in HEK293T cells individually or in combination. Lysates were immunoblotted to detect myc, V5 and HA epitopes. ( C ) Quantification of (A) and (B), where the scatter bar graph represents mean fold change ± SEM in the levels of myc-AP3B1, when co-expressed with native CK2α or inactive CK2α K68M, in the absence of IP6K1, normalized to the loading control. Data were analyzed using a one-sample t -test ( N =3). ( D ) Phosphorylation of AP3B1 by CK2. Myc-tagged AP3B1 immunoprecipitated from HEK293T cells was incubated with CK2 holoenzyme or GST-CK2α and radiolabeled [γ- 32 P]ATP in the absence or presence of 15 µM TBCA (CK2 inhibitor). DMSO was used as a vehicle control. Images show immunoblotting with a myc-tag antibody (left) and autoradiography to detect phosphorylation (right). Cells transfected with an empty-vector were used as a negative control. ( E ) Quantification of (D), where the scatter bar graph represents mean fold change ± SEM in the extent of phosphorylation of myc-AP3B1 in the presence or absence of TBCA. Data were analyzed using a one-sample t -test ( N =3). ( F ) Effect of TBCA treatment on cellular levels of AP3B1. Myc-tagged AP3B1 was expressed with or without HA-tagged CK2α in HEK293T cells, followed by treatment with 50 µM TBCA (16 h) or DMSO as a vehicle control. Lysates were immunoblotted to detect myc and HA epitopes, and tubulin as a loading control. ( G ) Quantification of (F), where the scatter bar graph represents mean fold change ± SEM in the levels of AP3B1 in TBCA treated compared with untreated cells, normalized to the loading control. Data were analyzed using a one-sample t -test ( N =3).

Article Snippet: Mouse full-length MYC cDNA with a C-terminal V5 tag was cloned in pCDNA3.1 as previously described [ ]. pZW12 CK2β was a gift from David Litchfield (Addgene plasmid 27088; GenBank ID NM_001320.7).

Techniques: Activity Assay, Control, Phospho-proteomics, Immunoprecipitation, Incubation, Western Blot, Autoradiography, Transfection, Plasmid Preparation, Negative Control

Journal: Bioscience Reports

Article Title: Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP 7

doi: 10.1042/BSR20240792

Figure Lengend Snippet: The α-subunit of protein kinase CK2 binds to AP3B1 and catalyzes its phosphorylation, priming it for 5-InsP 7 -mediated pyrophosphorylation. IP6K1 interacts with AP3B1 via its IDR-1 region and synthesizes 5-InsP 7 in the vicinity of the complex. A local increase in the concentration of 5-InsP 7 promotes the mass-action driven transfer of its β-phosphate moiety to pyrophosphorylate AP3B1.

Article Snippet: Mouse full-length MYC cDNA with a C-terminal V5 tag was cloned in pCDNA3.1 as previously described [ ]. pZW12 CK2β was a gift from David Litchfield (Addgene plasmid 27088; GenBank ID NM_001320.7).

Techniques: Phospho-proteomics, Concentration Assay