ck2 reaction buffer  (New England Biolabs)

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) GST-TopBP1 or GST-NEDD4 (0.65 pmol) was incubated with 11 pmol E2 and incubated at 4°C for 1 h with rotation. GST pulldowns followed by Western blotting for TopBP1 (top blot) and E2 (bottom blot) were then carried out. (B) The GST pulldown was repeated as in panel A following 1 h at 30°C with CK2 and controls. (C) Lambda phosphatase was added to the CK2 reaction mixture, and GST pulldown assays were carried out as in panel A. E2-specific antibody TVG261 (ab17185) was used for Western blotting in all three experiments (A to C). Figure S1A to S1C in the supplemental material summarizes quantitation of repeat experiments. In the input lanes, only E2 was added. The TopBP1 pulldown demonstrates equivalent levels of TopBP1 in each reaction mixture containing TopBP1.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Incubation, Western Blot, Quantitation Assay

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) siRNA knockdown of CK2α and/or CK2α′. Scr control siRNA was used in lanes 1 and 5. The top panels demonstrate the input proteins that were used in the immunoprecipitation (IP) with pS23-Ab (an antibody raised against an E2 peptide containing a phosphorylated serine 23). Please note the CK2α blot is independent of the other inputs but is run with the same protein extracts. The IP was blotted for E2 which is clearly detected in the Scr control (lane 5), but not in the CK2 knockdown cells (lanes 2 to 4). (B) The indicated cells were treated with DMSO (lanes 1 and 2) or the CK2 inhibitor CX4945 (lanes 3 and 4), and Western blotting details the levels of TopBP1 and E2 in the treated cells (top blots). Immunoprecipitation (IP) with TopBP1 demonstrates a pulldown of TopBP1 and E2 (lane 2) that is abrogated by CX4945 (lane 4) (middle blots). IP with pS23-Ab pulled down E2 in control cells (lane 2) that was abolished by CX4945 (lane 4). (C) CK2a siRNA knockdown (lane 4) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2α (lane 7). (D) CK2α′ knockdown (lane 3) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2α′ (lane 5). Please note that lanes 1 to 3 are from the same gel and the same exposure with a lane removed. (E) Staining of U2OS E2-WT (top panels) and U2OS E2-S23A (bottom panels) with pS23-Ab. Left-hand panels are antibody only, right-hand panels are antibody plus DAPI. There was no signal generated with secondary antibody only, and no signal detected in U2OS-Vec control when the primary antibody was included. HPV16 E2 B9 monoclonal antibody was used for Western blotting in panels A and B. E2-specific antibody TVG261 (ab17185) was used for Western blotting in panels C and D. Figure S2 A and B summarizes quantitation for repeat experiments of panels C and D, respectively. An asterisk indicates an antibody band.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Staining, Generated, Quantitation Assay

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) Top blots with lanes 1 to 3 are Western blots of extracts from the indicated stable N/Tert-1 cell lines. Bottom blots with lanes 4 to 6 are Western blots of a TopBP1 immunoprecipitation (IP) of the indicated extracts. TopBP1 co-IPs E2-WT but not E2-S23A. (B) The extracts in the top blots (Input) were immunoprecipitated with pS23-Ab, and E2 is pulled down by this antibody (bottom blot, lane 2). The CK2 inhibitor CX4945 abrogates this pulldown (lane 5). (C) The extracts from panel B were immunoprecipitated with TopBP1, and both E2-WT and E2-S23D co-IP with TopBP1 (lanes 2 and 3). Treatment with the CX4945 abrogates the interaction between TopBP1 and E2-WT (lane 6), but not E2-S23D (lane 5). (D) Organotypic raft cultures of N/Tert-1 (top panels) and N/Tert-1+HPV16 (bottom panels) were stained with pS23-Ab. There is no specific staining in N/Tert-1 cells, but there is clear staining in N/Tert-1+HPV16. (Left panels) pS23-Ab only, (right panels) pS23-Ab plus DAPI staining. An asterisk indicates an antibody band. (E) Organotypic raft cultures of N/Tert-1 (top panels) and N/Tert-1+HPV16 (bottom panels) stained with CK2 antibody. HPV16 E2 B9 monoclonal antibody was used for Western blotting in panels A to C.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: Agents used in N2a-based pharmacological screens.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques:

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: CK2 phosphorylates mammalian FMRP S499 in vitro. rFMRP S500 or S500D was incubated with or without recombinant CK2 for 30 min. Samples were resolved by SDS-PAGE and probed with pFMRP S499, tFMRP, or CK2a1 antibodies. Only rFMRP S499 incubated with CK2 showed a positive phosphosignal.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: In Vitro, Incubation, Recombinant, SDS Page

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: CK2 phosphorylates mammalian FMRP S499. A , Immunoblots for 3-h treatment of N2a cells with vehicle (DMSO), D4476 (25 µ m ), IC261 (20 µ m ), PHA-767491 (5 µ m ), DRB (50 µ m ), TBB (25 µ m ), or βARK (200 µ m ) followed by Western blot for protein indicated on the left. B , Quantification of pFMRP/tFMRP signal in A . ns, not significant (Kruskal–Wallis one-way ANOVA analysis [ H (8) = 4.56, p = 0.8034], n = 4). C , Immunoblot for 24-h treatment of N2a cells with the same agents listed in A . D , Quantification of pFMRP/tFMRP signal in C (Kruskal–Wallis one-way ANOVA [ H (8) = 7.239, p = 0.5111], n = 4, error bars = SEM). E , Baseline, untreated N2a cells were collected at time 0, and the remainder of the cells were treated with either DMSO or CX-4945 (5 or 1 µ m ) for 24 h. tFMRP and pFMRP S499 signals increased in DMSO-treated samples; however, only tFMRP increased in CX-treated samples, thereby causing a significant reduction in relative FMRP S499 phosphorylation. All immunoblot signals are from the same membrane; however, intervening lanes have been removed for clarity. F , Quantification of pFMRP S499 to tFMRP ratio from D ; one-way ANOVA, n = 4, error bars = SEM, * p < 0.05. G , HEK293 cells were collected at baseline (time 0) or treated for 24 h with DMSO or CX-4945. H , CX-4945 significantly reduced FMRP S499 phosphorylation compared with DMSO (one-tailed Mann–Whitney test, p = 0.0143). I , J , Mouse cortical neurons at 7 d in vitro treated with 1 µ m CX-4945 for 24 h exhibited a significant reduction in FMRP S499 phosphorylation compared with DMSO-treated neurons (one-tailed Mann–Whitney test, p = 0.05).

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: Western Blot, One-tailed Test, MANN-WHITNEY, In Vitro

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: FMRP S499 rephosphorylation kinetics after CX-4945 treatment and washout mirrors a known CK2 target. A , N2a cells were collected either at baseline or 24 h after DMSO or CX-4945 treatment. Additional samples were collected after 24-h treatment and washout of DMSO or CX-4945 for different time periods. B , Quantification of [p/t]FMRP or [p/t]AKT compared with baseline. Changes in ratios were quantified by two-way ANOVA followed by Dunnett’s multiple comparisons post hoc test, n = 4 per data point, error bars = SEM. **** p < 0.0001; *** p < 0.001; * p < 0.05.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques:

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: Alternative model for FMRP phosphorylation and regulation of translation. FMRP is first phosphorylated by CK2 on S499. FMRP S499 phosphorylation is permissive for secondary phosphorylation of FMRP on serine/threonine residues, presumably downstream of mGluR-I, by unknown kinases. Secondary phosphorylation of FMRP is counteracted by PP2A-mediated dephosphorylation. The dotted green and red lines are provisional and indicate that the relationship between FMRP’s phosphorylation status and protein translation is unknown.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: De-Phosphorylation Assay