Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Identification of the acetylation of the K376 residue of G3BP1. (A) Mass spectrometric identification of the acetylated and intact G3BP1 peptide INSGGK376LPNFGFVVFDDSEPVQK. (B and C) FLAG-tagged WT or K376Q G3BP1 or FLAG vector control (Vec) were transfected into 293T (B) or NSC-34 (C) cells. A day later, the cells were treated with the deacetylase inhibitors TSA, sodium butyrate, and nicotinamide as indicated overnight, followed by FLAG immunoprecipitation and immunoblotting (IB) with the indicated antibodies. Representative results of at least three independent experiments are shown.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Plasmid Preparation, Transfection, Histone Deacetylase Assay, Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Testing the acetylated-K376 G3BP1-specific affinity-purified rabbit polyclonal antibody. (A) 293T and G3BP1-null 293T cells were left untreated or were treated with the deacetylase inhibitors TSA, sodium butyrate, and nicotinamide overnight before lysate preparation. Immunoblotting of the extracts was performed with the indicated antibodies. (B) G3BP1-null 293T cells were transfected with FLAG-tagged G3BP1, G3BP2A, and G3BP2B expression constructs or FLAG vector control. A day later, the cells were treated with the deacetylase inhibitors TSA, sodium butyrate, and nicotinamide overnight before lysate preparation, followed by FLAG immunoprecipitation and immunoblotting with the indicated antibodies. Representative results from at least three independent experiments are shown. (C) The anti-acetylated K376 G3BP1 antibody (Ac-G3BP1) was unable to detect the acetylated G3BP1 band in DACi-treated 293T lysate when the antibody was blocked by preincubation with the immunizing peptide at a 1-μg/ml concentration for 1 h at room temperature.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Affinity Purification, Histone Deacetylase Assay, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Immunoprecipitation, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: G3BP1 K376 acetylation is regulated by HDAC6 and CBP. (A) Hyperacetylated FLAG-tagged G3BP1 was immunoprecipitated from deacetylase inhibitor-treated 293T cells under native conditions and subsequently deacetylated in vitro with purified human HDAC6. (B) Endogenous G3BP1 is hyperacetylated at the K376 position in HDAC6-null MEF cells. The specific HDAC6 band is marked by an asterisk. (C) Overexpression of CBP resulted in hyperacetylation of the cotransfected FLAG-tagged WT but not K376Q mutant G3BP1. FLAG immunoprecipitation followed by immunoblotting with the indicated antibodies is shown. Representative results of at least three independent experiments are shown. The numbers under the respective panels represent the quantification of the FLAG-G3BP1 signal in the immunoprecipitations and the FLAG-G3BP1 signal normalized to the actin levels in the total extracts (Ext).

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Immunoprecipitation, Histone Deacetylase Assay, In Vitro, Purification, Over Expression, Mutagenesis, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Acetylation of the K376 residue and the K376Q acetylation-mimicking mutation impair RNA binding in RNA-IP experiments. (A) The domain structure of G3BP1. (B and C) The indicated FLAG-tagged G3BP1 expression constructs were transfected into 293T (B) or NSC-34 (C) cells. Two days later, the cells were lysed and RNA immunoprecipitation experiments were performed. The c-myc and tau mRNA amounts were normalized with the FLAG-G3BP1 levels in the FLAG-G3BP1 immunoprecipitations and with the RPL13A mRNA levels in the total extracts. Averages ± SD from three repetitions are shown. (D) In vitro RNA-IP of the c-myc mRNA from total 293T RNA with nonacetylated or K376-acetylated recombinant FLAG-G3BP1-6×His bait. Averages ± SD from three repetitions are shown. *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P < 0.001.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Mutagenesis, RNA Binding Assay, Expressing, Construct, Transfection, Immunoprecipitation, In Vitro, Recombinant

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: K376Q acetylation-mimicking mutation impaired the interaction of G3BP1 with PABP1 but not with caprin-1 or USP10. (A) The indicated FLAG-tagged G3BP1 expression constructs were transfected into 293T cells. Two days later, the cells were lysed and FLAG immunoprecipitations were performed, followed by immunoblotting with the indicated antibodies. (B to D) Quantification of experiments shown in panel A from three independent repetitions. Ext, total cell extracts. **, 0.01 > P > 0.001.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Mutagenesis, Expressing, Construct, Transfection, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Effect of CBP/p300 inhibition on G3BP1 stress granule dynamics. (A) 293T cells were pretreated with deacetylase inhibitors and the indicated concentrations of the highly selective CBP/p300 inhibitor A-485 (6387; Tocris) as shown for 1 h in the medium, followed by 1 h of SG induction with 0.5 mM sodium arsenite and 5 h of recovery in the respective fresh medium with DACi and A-485. The control cells were treated with sodium arsenite only. The cell lysates were subjected to immunoblotting with the indicated antibodies. (B) Schematic of the treatment of 293T cells with A-485 (10 μM) or vehicle (DMSO) and sodium arsenite (0.5 mM). (C) Representative confocal microscopic images of G3BP1 and TIA-1 immunofluorescence of 293T cells treated with sodium arsenite for 1 h, followed by recovery in the respective fresh media for 3 h. The nuclei were visualized with DAPI stain. Scale bars, 20 μm. (D and E) The number ± SD of G3BP1 SGs per cell (D) and the average size ± SD of G3BP1 SGs (E), as quantified in four independent experiments. *, 0.05 > P > 0.01; **, 0.01 > P > 0.001.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Inhibition, Histone Deacetylase Assay, Western Blot, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: K376Q acetylation-mimicking mutation and the F380L/F382L RNA binding-deficient mutation impair SG formation. (A) The G3BP1-null 293T derivative mCherry-G3BP1 WT, K376Q, and F380L/F382L knock-in cell lines had G3BP1 levels comparable to that of 293T cells and each other, as shown in immunoblotting with the indicated antibodies. Symbols: #, the expected mCherry-tagged G3BP1 band; ##, mCherry-G3BP1 fusion protein with likely partially processed mCherry tag. See the text for details. (B) Representative confocal microscopic images of mCherry-G3BP1 fluorescence and TIA-1 immunofluorescence of the varied knock-in cell lines after 30 min of treatment with 0.5 mM sodium arsenite. The nuclei were visualized with DAPI stain. Scale bars, 20 μm. (C) The number ± SD of G3BP1 SGs per cell and the average size ± SD of G3BP1 SGs, as quantified in four independent experiments. The F380L/F382L error bars are shown semitransparent on the right to allow for distinction between the WT and K376Q error bars at 30 min. Significance compared to values for the WT is indicated by asterisks: *, 0.05> P > 0.01; **, 0.01 > P > 0.001; ***, P < 0.001. Rec, recovery.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Mutagenesis, RNA Binding Assay, Knock-In, Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Time course of G3BP1 acetylation during SG induction and disassembly. (A) Schematic of the treatment of 293T cells with deacetylase inhibitors and sodium arsenite (0.5 mM). The control cells were left untreated. (B) Immunoblotting of the respective cell lysates treated as described for panel A with the indicated antibodies. (C) Quantification of the specific K376 acetylation of G3BP1, normalized to the DACi-treated, sodium arsenite-untreated condition. The averages ± SD from three experiments are shown. (D) Representative confocal microscopic images of G3BP1 immunofluorescence (white pseudocoloring) at various time points as shown in panel A. The nuclei were visualized with DAPI stain. Scale bars, 20 μm. (E) Quantification of the number ± SD of G3BP1 granules per cell, as shown in panel D, from four independent experiments. **, 0.01 > P > 0.001; ***, P < 0.001. Ars, arsenite; Rec, recovery.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Histone Deacetylase Assay, Western Blot, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Regulation of the interactions of G3BP1 with other SG proteins under stress conditions. (A) The FLAG-tagged WT G3BP1 expression construct or vector control was transfected into 293T cells. Two days later, the cells were either left untreated or were treated for 30 min or 1 h with 0.5 mM sodium arsenite in the medium, followed by FLAG immunoprecipitation and immunoblotting with the indicated antibodies. Representative images of three repetitions are shown. (B) The FLAG-tagged WT, K376Q, or K376R G3BP1 expression construct or vector control was transfected into 293T cells. Two days later, the cells expressing FLAG-G3BP1 were treated for 1 h with 0.5 mM sodium arsenite in the medium, followed by 5 h of recovery without sodium arsenite in the presence of deacetylase inhibitors (DACi). The cells were lysed and FLAG immunoprecipitations were performed, followed by immunoblotting with the indicated antibodies. Under the top panel, the relative quantification of the coprecipitated PABP1 protein is shown from three repetitions. Only the coprecipitation of PABP1 with WT or K376Q G3BP1 was statistically significantly different (see the text for details). Ext, total cell extracts.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Expressing, Construct, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Histone Deacetylase Assay

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: K376-acetylated G3BP1 localizes primarily outside SGs. Representative confocal microscopic images of mCherry-G3BP1 fluorescence and the K376-acetylated G3BP1 PLA foci (Ac-K376 PLA). The nuclei were visualized with DAPI stain. Acetylated G3BP1/total G3BP1 proximity ligation assay was performed on G3BP1-null 293T-derived cells stably expressing mCherry-WT G3BP1. The cells were pretreated with deacetylase inhibitors for 1 h, followed by SG induction with 0.5 mM sodium arsenite for 1 h and 1 or 3 h of recovery in fresh medium containing deacetylase inhibitors. The acetylated G3BP1 PLA foci were often found in the periphery of the disassembling SGs. The insets show magnifications of the boxed areas. Scale bars, 5 μm.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Fluorescence, Staining, Proximity Ligation Assay, Derivative Assay, Stable Transfection, Expressing, Histone Deacetylase Assay

Journal: Molecular and Cellular Biology

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

doi: 10.1128/MCB.00052-19

Figure Lengend Snippet: Proposed model of the regulation of stress granule dynamics through G3BP1 K376 acetylation by HDAC6 and CBP/p300.

Article Snippet: The rabbit polyclonal anti-K376-acetylated G3BP1 antibody was generated by immunizing rabbits with the acetylated peptide antigen conjugated to KLH (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: