Structured Review

Millipore anti flag m2 affinity gel
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Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

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1) Product Images from "Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior"

Article Title: Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkae093

Key resources table
Figure Legend Snippet: Key resources table

Techniques Used: Clone Assay, Sequencing, Multiplex Assay, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Methylation, Software


Structured Review

Millipore anti flag m2 affinity gel
Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

Millipore anti flag m2 affinity gel
Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

Millipore ez view red anti flag m2 affinity gel

Ez View Red Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ez view red anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
ez view red anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

Images

1) Product Images from "Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity"

Article Title: Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity

Journal: Cell reports

doi: 10.1016/j.celrep.2024.113965


Figure Legend Snippet:

Techniques Used: Virus, Recombinant, Protease Inhibitor, Electron Microscopy, Lysis, Extraction, Staining, Mutagenesis, Software


Structured Review

Millipore anti flag m2 affinity gel
A Schematic representation of CCR4-NOT. Yellow circles indicate points of contact with UNK IDR identified in this study. The numbering of contacts indicates SLiM 1 or SLiM 2 binding sites. Adapted from Raisch et al. . under permission provided by a Creative Commons Attribution 4.0 International License. B Pull-down assays with recombinant UNK FULL tagged with the StrepII (Strep) affinity tag upon incubation with different CCR4-NOT modules ( n = 3). C As in ( B ) but with mutants of UNK IDR constructs fused to MBP and Strep after incubation with the NOT9 or the NOT module ( n = 3). D Twenty-five AlphaFold predictions of interfaces of UNK IDR interacting with the NOT9 module. The predictions are aligned on the CNOT9/CNOT1 heterodimer . The region of UNK IDR where the predictions converged is in dark blue. E The converged region of UNK IDR bound on the concave surface of CNOT9/CNOT1. F The same 25 predictions as in ( D ) but oriented to show the tryptophan (W)-binding pockets of CNOT9. G All 25 predictions of the converged region of UNK IDR close to the W-binding pockets of CNOT9. H Twenty-five AlphaFold predictions of UNK IDR interacting with the NOT module. The predictions are aligned on the CNOT1/CNOT2/CNOT3 heterotrimer . The region of UNK IDR where predictions converged is in dark blue. I The converged region of UNK IDR bound on the surface of CNOT1. J Pull-down of WT or M1-M3 mutants of the NOT9 module by MBP-UNK IDR -Strep. Residues in CNOT9 mutated to alanines in M1 (Y203 and R244) line the W-pocket 1, and those mutated in <t>M2</t> (R205 and H208) line the W-pocket 2 . All residues (Y203, R205, H208, and R244) were mutated in M3 ( n = 2). K Pull-down of the WT or M3 NOT9 module or the NOT module by WT MBP-UNK IDR -Strep or its mutant with key residues in SLiM 1 (V511, I515, L522) substituted with glutamic acid. In ( E ), ( G ), and ( I ), the C-alpha atoms of the key interacting residues are shown as yellow spheres. The sequences of the converged region are shown with the key interacting residues in yellow.
Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

Images

1) Product Images from "Regulation by the RNA-binding protein Unkempt at its effector interface"

Article Title: Regulation by the RNA-binding protein Unkempt at its effector interface

Journal: Nature Communications

doi: 10.1038/s41467-024-47449-4

A Schematic representation of CCR4-NOT. Yellow circles indicate points of contact with UNK IDR identified in this study. The numbering of contacts indicates SLiM 1 or SLiM 2 binding sites. Adapted from Raisch et al. . under permission provided by a Creative Commons Attribution 4.0 International License. B Pull-down assays with recombinant UNK FULL tagged with the StrepII (Strep) affinity tag upon incubation with different CCR4-NOT modules ( n = 3). C As in ( B ) but with mutants of UNK IDR constructs fused to MBP and Strep after incubation with the NOT9 or the NOT module ( n = 3). D Twenty-five AlphaFold predictions of interfaces of UNK IDR interacting with the NOT9 module. The predictions are aligned on the CNOT9/CNOT1 heterodimer . The region of UNK IDR where the predictions converged is in dark blue. E The converged region of UNK IDR bound on the concave surface of CNOT9/CNOT1. F The same 25 predictions as in ( D ) but oriented to show the tryptophan (W)-binding pockets of CNOT9. G All 25 predictions of the converged region of UNK IDR close to the W-binding pockets of CNOT9. H Twenty-five AlphaFold predictions of UNK IDR interacting with the NOT module. The predictions are aligned on the CNOT1/CNOT2/CNOT3 heterotrimer . The region of UNK IDR where predictions converged is in dark blue. I The converged region of UNK IDR bound on the surface of CNOT1. J Pull-down of WT or M1-M3 mutants of the NOT9 module by MBP-UNK IDR -Strep. Residues in CNOT9 mutated to alanines in M1 (Y203 and R244) line the W-pocket 1, and those mutated in M2 (R205 and H208) line the W-pocket 2 . All residues (Y203, R205, H208, and R244) were mutated in M3 ( n = 2). K Pull-down of the WT or M3 NOT9 module or the NOT module by WT MBP-UNK IDR -Strep or its mutant with key residues in SLiM 1 (V511, I515, L522) substituted with glutamic acid. In ( E ), ( G ), and ( I ), the C-alpha atoms of the key interacting residues are shown as yellow spheres. The sequences of the converged region are shown with the key interacting residues in yellow.
Figure Legend Snippet: A Schematic representation of CCR4-NOT. Yellow circles indicate points of contact with UNK IDR identified in this study. The numbering of contacts indicates SLiM 1 or SLiM 2 binding sites. Adapted from Raisch et al. . under permission provided by a Creative Commons Attribution 4.0 International License. B Pull-down assays with recombinant UNK FULL tagged with the StrepII (Strep) affinity tag upon incubation with different CCR4-NOT modules ( n = 3). C As in ( B ) but with mutants of UNK IDR constructs fused to MBP and Strep after incubation with the NOT9 or the NOT module ( n = 3). D Twenty-five AlphaFold predictions of interfaces of UNK IDR interacting with the NOT9 module. The predictions are aligned on the CNOT9/CNOT1 heterodimer . The region of UNK IDR where the predictions converged is in dark blue. E The converged region of UNK IDR bound on the concave surface of CNOT9/CNOT1. F The same 25 predictions as in ( D ) but oriented to show the tryptophan (W)-binding pockets of CNOT9. G All 25 predictions of the converged region of UNK IDR close to the W-binding pockets of CNOT9. H Twenty-five AlphaFold predictions of UNK IDR interacting with the NOT module. The predictions are aligned on the CNOT1/CNOT2/CNOT3 heterotrimer . The region of UNK IDR where predictions converged is in dark blue. I The converged region of UNK IDR bound on the surface of CNOT1. J Pull-down of WT or M1-M3 mutants of the NOT9 module by MBP-UNK IDR -Strep. Residues in CNOT9 mutated to alanines in M1 (Y203 and R244) line the W-pocket 1, and those mutated in M2 (R205 and H208) line the W-pocket 2 . All residues (Y203, R205, H208, and R244) were mutated in M3 ( n = 2). K Pull-down of the WT or M3 NOT9 module or the NOT module by WT MBP-UNK IDR -Strep or its mutant with key residues in SLiM 1 (V511, I515, L522) substituted with glutamic acid. In ( E ), ( G ), and ( I ), the C-alpha atoms of the key interacting residues are shown as yellow spheres. The sequences of the converged region are shown with the key interacting residues in yellow.

Techniques Used: Binding Assay, Recombinant, Incubation, Construct, Mutagenesis


Structured Review

Millipore anti flag m2 affinity gel
Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 affinity gel - by Bioz Stars, 2024-05
86/100 stars

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ez view red anti flag m2 affinity gel millipore sigma  (Millipore)

 
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    Structured Review

    Millipore ez view red anti flag m2 affinity gel millipore sigma
    Ez View Red Anti Flag M2 Affinity Gel Millipore Sigma, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez view red anti flag m2 affinity gel millipore sigma/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ez view red anti flag m2 affinity gel millipore sigma - by Bioz Stars, 2024-05
    86/100 stars

    Images


    Structured Review

    Millipore anti flag m2 affinity gel
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag m2 affinity gel - by Bioz Stars, 2024-05
    86/100 stars

    Images


    Structured Review

    Millipore anti flag m2 affinity gel
    List of antibodies used in this study
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag m2 affinity gel - by Bioz Stars, 2024-05
    86/100 stars

    Images

    1) Product Images from "Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways"

    Article Title: Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae002

    List of antibodies used in this study
    Figure Legend Snippet: List of antibodies used in this study

    Techniques Used:

    Validation of K142 and K226 acetylation sites using custom-generated anti-Ac (K142 or K226) specific antibodies (Ab) in stable cell lines. ( A ) PNKP was IP’d using the anti-AcK142 Ab from the chromatin fraction of GO-treated WT (lane 1) and K142R mutant (lane 2) PNKP expressing cells and probed with the anti-FLAG Ab (upper panel). Lower panel: Input shows similar ectopic expression of PNKP-FLAG in the cell lines, detected with the anti-FLAG Ab. ( B ) Indirect immunofluorescence was conducted to detect nuclear localization of AcK142 PNKP in GO-treated WT vs. mutant (K142R and K226R) cell lines. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( C ) PNKP was IP’d using the anti-AcK226 Ab from the chromatin fraction of mock (lanes 1, 2) or Bleo-treated (lanes 3, 4) WT (lanes 1, 3) and K226R mutant (lanes 2, 4) PNKP expressing cells and probed with anti-FLAG Ab (Upper Panel). Lower panel shows the inputs, indicating similar ectopic expression of PNKP-FLAG in the cell lines detected with anti-FLAG Ab. ( D ) Mock- or Bleo-treated WT and mutant K226R PNKP expressing cells were stained with an anti-AcK226 Ab. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( E ) Indirect immunofluorescence was performed to assess the status of AcK226 PNKP in mock vs. Bleo-treated WT-PNKP expressing cells at 3 h, 6 h and 9 h post-Bleo treatment. Scale bar: 40 μm. All the immunofluorescence experiments were performed following depletion of endogenous PNKP by 3′-UTR specific siRNA.
    Figure Legend Snippet: Validation of K142 and K226 acetylation sites using custom-generated anti-Ac (K142 or K226) specific antibodies (Ab) in stable cell lines. ( A ) PNKP was IP’d using the anti-AcK142 Ab from the chromatin fraction of GO-treated WT (lane 1) and K142R mutant (lane 2) PNKP expressing cells and probed with the anti-FLAG Ab (upper panel). Lower panel: Input shows similar ectopic expression of PNKP-FLAG in the cell lines, detected with the anti-FLAG Ab. ( B ) Indirect immunofluorescence was conducted to detect nuclear localization of AcK142 PNKP in GO-treated WT vs. mutant (K142R and K226R) cell lines. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( C ) PNKP was IP’d using the anti-AcK226 Ab from the chromatin fraction of mock (lanes 1, 2) or Bleo-treated (lanes 3, 4) WT (lanes 1, 3) and K226R mutant (lanes 2, 4) PNKP expressing cells and probed with anti-FLAG Ab (Upper Panel). Lower panel shows the inputs, indicating similar ectopic expression of PNKP-FLAG in the cell lines detected with anti-FLAG Ab. ( D ) Mock- or Bleo-treated WT and mutant K226R PNKP expressing cells were stained with an anti-AcK226 Ab. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( E ) Indirect immunofluorescence was performed to assess the status of AcK226 PNKP in mock vs. Bleo-treated WT-PNKP expressing cells at 3 h, 6 h and 9 h post-Bleo treatment. Scale bar: 40 μm. All the immunofluorescence experiments were performed following depletion of endogenous PNKP by 3′-UTR specific siRNA.

    Techniques Used: Generated, Stable Transfection, Mutagenesis, Expressing, Immunofluorescence, Staining

    Assessment of the effect of acetylation on PNKP’s 3′-phosphatase and 5′-kinase activities. ( A ) The Western blot shows the expression of γH2AX (upper panel) in the chromatin fraction of WT (lanes 1, 2), K142R (lanes 3, 4), K226R (lanes 5, 6) and K142R/226R (lanes 7, 8) PNKP expressing cells post mock (–) or Bleo (+) treatment. HDAC2 is used as the loading control for the chromatin fraction (lower panel). ( B ) IP’d and purified FLAG-PNKP from WT and mutant PNKP expressing cells (± Bleo) was immunoblotted with anti-FLAG Ab (upper panel). 10 μg of the IP’d chromatin fraction from WT and K-R mutant PNKP expressing cells was used as input (lower panel). ( C ) A schematic representation of the PNKP's 3′-phosphatase assay. ( D ) The 3′-phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lanes 2–3), K142R (lane 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) PNKP-expressing cells post mock (–) or Bleo (+) treatment. Lane 1: No protein (NP), substrate only. Lower panel represents the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, * P < 0.05 between mock (–) and Bleo (+) treatment in WT and K142R; ns: non-significant, P > 0.05). ( E ) The phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lane 2), K142Q (lane 3), K226Q (lane 4) and K142Q/226Q (lane 5). Lane 1: no protein (NP), substrate only. Lower panel shows the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, ns: non-significant, P > 0.05). ( F ) 5′-kinase assay was conducted with IP’d FLAG-PNKP from WT (lane 2–3), K142R (lanes 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) post mock (–) or Bleo (+) treatment. Lane 10: radiolabeled 32 nt marker (M). Lane 1: No protein (NP), substrate only. The quantitation of the products (% phosphorylated product) is represented in the bar diagram (Error bars show ± SD of the mean; n = 3, ns = non-significant, P > 0.05). In each case of (D–F), S: substrate and P: product.
    Figure Legend Snippet: Assessment of the effect of acetylation on PNKP’s 3′-phosphatase and 5′-kinase activities. ( A ) The Western blot shows the expression of γH2AX (upper panel) in the chromatin fraction of WT (lanes 1, 2), K142R (lanes 3, 4), K226R (lanes 5, 6) and K142R/226R (lanes 7, 8) PNKP expressing cells post mock (–) or Bleo (+) treatment. HDAC2 is used as the loading control for the chromatin fraction (lower panel). ( B ) IP’d and purified FLAG-PNKP from WT and mutant PNKP expressing cells (± Bleo) was immunoblotted with anti-FLAG Ab (upper panel). 10 μg of the IP’d chromatin fraction from WT and K-R mutant PNKP expressing cells was used as input (lower panel). ( C ) A schematic representation of the PNKP's 3′-phosphatase assay. ( D ) The 3′-phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lanes 2–3), K142R (lane 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) PNKP-expressing cells post mock (–) or Bleo (+) treatment. Lane 1: No protein (NP), substrate only. Lower panel represents the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, * P < 0.05 between mock (–) and Bleo (+) treatment in WT and K142R; ns: non-significant, P > 0.05). ( E ) The phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lane 2), K142Q (lane 3), K226Q (lane 4) and K142Q/226Q (lane 5). Lane 1: no protein (NP), substrate only. Lower panel shows the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, ns: non-significant, P > 0.05). ( F ) 5′-kinase assay was conducted with IP’d FLAG-PNKP from WT (lane 2–3), K142R (lanes 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) post mock (–) or Bleo (+) treatment. Lane 10: radiolabeled 32 nt marker (M). Lane 1: No protein (NP), substrate only. The quantitation of the products (% phosphorylated product) is represented in the bar diagram (Error bars show ± SD of the mean; n = 3, ns = non-significant, P > 0.05). In each case of (D–F), S: substrate and P: product.

    Techniques Used: Western Blot, Expressing, Purification, Mutagenesis, Phosphatase Assay, Quantitation Assay, Kinase Assay, Marker


    Structured Review

    Millipore anti flag m2 affinity gel
    List of antibodies used in this study
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag m2 affinity gel/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag m2 affinity gel - by Bioz Stars, 2024-05
    86/100 stars

    Images

    1) Product Images from "Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways"

    Article Title: Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae002

    List of antibodies used in this study
    Figure Legend Snippet: List of antibodies used in this study

    Techniques Used:

    Validation of K142 and K226 acetylation sites using custom-generated anti-Ac (K142 or K226) specific antibodies (Ab) in stable cell lines. ( A ) PNKP was IP’d using the anti-AcK142 Ab from the chromatin fraction of GO-treated WT (lane 1) and K142R mutant (lane 2) PNKP expressing cells and probed with the anti-FLAG Ab (upper panel). Lower panel: Input shows similar ectopic expression of PNKP-FLAG in the cell lines, detected with the anti-FLAG Ab. ( B ) Indirect immunofluorescence was conducted to detect nuclear localization of AcK142 PNKP in GO-treated WT vs. mutant (K142R and K226R) cell lines. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( C ) PNKP was IP’d using the anti-AcK226 Ab from the chromatin fraction of mock (lanes 1, 2) or Bleo-treated (lanes 3, 4) WT (lanes 1, 3) and K226R mutant (lanes 2, 4) PNKP expressing cells and probed with anti-FLAG Ab (Upper Panel). Lower panel shows the inputs, indicating similar ectopic expression of PNKP-FLAG in the cell lines detected with anti-FLAG Ab. ( D ) Mock- or Bleo-treated WT and mutant K226R PNKP expressing cells were stained with an anti-AcK226 Ab. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( E ) Indirect immunofluorescence was performed to assess the status of AcK226 PNKP in mock vs. Bleo-treated WT-PNKP expressing cells at 3 h, 6 h and 9 h post-Bleo treatment. Scale bar: 40 μm. All the immunofluorescence experiments were performed following depletion of endogenous PNKP by 3′-UTR specific siRNA.
    Figure Legend Snippet: Validation of K142 and K226 acetylation sites using custom-generated anti-Ac (K142 or K226) specific antibodies (Ab) in stable cell lines. ( A ) PNKP was IP’d using the anti-AcK142 Ab from the chromatin fraction of GO-treated WT (lane 1) and K142R mutant (lane 2) PNKP expressing cells and probed with the anti-FLAG Ab (upper panel). Lower panel: Input shows similar ectopic expression of PNKP-FLAG in the cell lines, detected with the anti-FLAG Ab. ( B ) Indirect immunofluorescence was conducted to detect nuclear localization of AcK142 PNKP in GO-treated WT vs. mutant (K142R and K226R) cell lines. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( C ) PNKP was IP’d using the anti-AcK226 Ab from the chromatin fraction of mock (lanes 1, 2) or Bleo-treated (lanes 3, 4) WT (lanes 1, 3) and K226R mutant (lanes 2, 4) PNKP expressing cells and probed with anti-FLAG Ab (Upper Panel). Lower panel shows the inputs, indicating similar ectopic expression of PNKP-FLAG in the cell lines detected with anti-FLAG Ab. ( D ) Mock- or Bleo-treated WT and mutant K226R PNKP expressing cells were stained with an anti-AcK226 Ab. Nuclei were counterstained with DAPI. Scale bar: 40 μm. ( E ) Indirect immunofluorescence was performed to assess the status of AcK226 PNKP in mock vs. Bleo-treated WT-PNKP expressing cells at 3 h, 6 h and 9 h post-Bleo treatment. Scale bar: 40 μm. All the immunofluorescence experiments were performed following depletion of endogenous PNKP by 3′-UTR specific siRNA.

    Techniques Used: Generated, Stable Transfection, Mutagenesis, Expressing, Immunofluorescence, Staining

    Assessment of the effect of acetylation on PNKP’s 3′-phosphatase and 5′-kinase activities. ( A ) The Western blot shows the expression of γH2AX (upper panel) in the chromatin fraction of WT (lanes 1, 2), K142R (lanes 3, 4), K226R (lanes 5, 6) and K142R/226R (lanes 7, 8) PNKP expressing cells post mock (–) or Bleo (+) treatment. HDAC2 is used as the loading control for the chromatin fraction (lower panel). ( B ) IP’d and purified FLAG-PNKP from WT and mutant PNKP expressing cells (± Bleo) was immunoblotted with anti-FLAG Ab (upper panel). 10 μg of the IP’d chromatin fraction from WT and K-R mutant PNKP expressing cells was used as input (lower panel). ( C ) A schematic representation of the PNKP's 3′-phosphatase assay. ( D ) The 3′-phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lanes 2–3), K142R (lane 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) PNKP-expressing cells post mock (–) or Bleo (+) treatment. Lane 1: No protein (NP), substrate only. Lower panel represents the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, * P < 0.05 between mock (–) and Bleo (+) treatment in WT and K142R; ns: non-significant, P > 0.05). ( E ) The phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lane 2), K142Q (lane 3), K226Q (lane 4) and K142Q/226Q (lane 5). Lane 1: no protein (NP), substrate only. Lower panel shows the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, ns: non-significant, P > 0.05). ( F ) 5′-kinase assay was conducted with IP’d FLAG-PNKP from WT (lane 2–3), K142R (lanes 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) post mock (–) or Bleo (+) treatment. Lane 10: radiolabeled 32 nt marker (M). Lane 1: No protein (NP), substrate only. The quantitation of the products (% phosphorylated product) is represented in the bar diagram (Error bars show ± SD of the mean; n = 3, ns = non-significant, P > 0.05). In each case of (D–F), S: substrate and P: product.
    Figure Legend Snippet: Assessment of the effect of acetylation on PNKP’s 3′-phosphatase and 5′-kinase activities. ( A ) The Western blot shows the expression of γH2AX (upper panel) in the chromatin fraction of WT (lanes 1, 2), K142R (lanes 3, 4), K226R (lanes 5, 6) and K142R/226R (lanes 7, 8) PNKP expressing cells post mock (–) or Bleo (+) treatment. HDAC2 is used as the loading control for the chromatin fraction (lower panel). ( B ) IP’d and purified FLAG-PNKP from WT and mutant PNKP expressing cells (± Bleo) was immunoblotted with anti-FLAG Ab (upper panel). 10 μg of the IP’d chromatin fraction from WT and K-R mutant PNKP expressing cells was used as input (lower panel). ( C ) A schematic representation of the PNKP's 3′-phosphatase assay. ( D ) The 3′-phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lanes 2–3), K142R (lane 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) PNKP-expressing cells post mock (–) or Bleo (+) treatment. Lane 1: No protein (NP), substrate only. Lower panel represents the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, * P < 0.05 between mock (–) and Bleo (+) treatment in WT and K142R; ns: non-significant, P > 0.05). ( E ) The phosphatase assay was performed with immuno-purified FLAG-PNKP from WT (lane 2), K142Q (lane 3), K226Q (lane 4) and K142Q/226Q (lane 5). Lane 1: no protein (NP), substrate only. Lower panel shows the quantitation of the products (% of released phosphate) (Error bars show ± SD of the mean; n = 3, ns: non-significant, P > 0.05). ( F ) 5′-kinase assay was conducted with IP’d FLAG-PNKP from WT (lane 2–3), K142R (lanes 4–5), K226R (lane 6–7) and K142R/226R (lanes 8–9) post mock (–) or Bleo (+) treatment. Lane 10: radiolabeled 32 nt marker (M). Lane 1: No protein (NP), substrate only. The quantitation of the products (% phosphorylated product) is represented in the bar diagram (Error bars show ± SD of the mean; n = 3, ns = non-significant, P > 0.05). In each case of (D–F), S: substrate and P: product.

    Techniques Used: Western Blot, Expressing, Purification, Mutagenesis, Phosphatase Assay, Quantitation Assay, Kinase Assay, Marker

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    Millipore anti flag m2 affinity gel
    Key resources table
    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Key resources table

    Journal: Nucleic Acids Research

    Article Title: Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior

    doi: 10.1093/nar/gkae093

    Figure Lengend Snippet: Key resources table

    Article Snippet: The supernatants were incubated with anti-Flag M2 Affinity Gel (A2220, Millipore) or anti-IgG Agarose (A0919, Sigma-Aldrich) as a control at 4°C overnight.

    Techniques: Clone Assay, Sequencing, Multiplex Assay, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Methylation, Software

    Journal: Cell reports

    Article Title: Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity

    doi: 10.1016/j.celrep.2024.113965

    Figure Lengend Snippet:

    Article Snippet: EZ view Red Anti-Flag M2 affinity gel , Millipore Sigma , Cat# F2426.

    Techniques: Virus, Recombinant, Protease Inhibitor, Electron Microscopy, Lysis, Extraction, Staining, Mutagenesis, Software