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Bethyl endogenous immunoprecipitation

2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to <t>immunoprecipitation</t> (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: .

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1) Product Images from "DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses"

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20251796

Figure Legend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to immunoprecipitation (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: .

Techniques Used: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding Assay

2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B. Whole-cell extracts (WCEs) from T98G cells were used as input for IPs using mock IgG and DNA-PKcs–specific antibodies prior to incubation with 2′3′-cGAMP and detection of bound 2′3′-cGAMP. (B) WB analysis of DNA-PKcs IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) Graph represents mean (±SEM; n = 3 independent experiment) 2′3′-cGAMP levels as measured in mock and DNA-PK–specific IP performed as in A. Statistical significance was calculated by two-tailed Student t test. (D) Silver staining was conducted on recombinant DNA-PKcs used in for immunoprecipitation experiments. Representative gel of three independent experiments. (E) WB analysis of TSA, conducted on WCE from THP-1 cells incubated with or without 2′3′-cGAMP or in presence or absence of NU7441. Immunoblot was performed using DNA-PKcs–, STING-, and HSP90-specific antibodies. Representative WB of three independent experiments. (F) Heatmap representation of the relative band intensities quantified from three independent experiments performed as in E. (G) Molecular modelling and docking study of 2′3′-cGAMP into DNA-PKcs. Human DNA-PKcs in ribbon representation with 2′3′-cGAMP docked in its catalytic site. (H) The docking conformation of 2′3′-cGAMP (in red spacefill representation) into the catalytic site of DNA-PKcs in the proximity of the catalytic residues (in ball and stick representation). (I) The docked conformation adopted by 2′3′-cGAMP onto the catalytic site of DNA-PKcs upon the MDSs. (J) The 2D molecular interactions diagram of 2′3′-cGAMP with the catalytic residues of DNA-PKcs. (K) Molecular modelling of ATM and ATR. DNA-PKcs superposed to the models of ATM and ATR (in red, blue, and yellow ribbon representations, respectively). (L) Close-up of the superposed active sites of ATM, ATR, and DNA-PKcs. Each of the three kinases has significant conformational differences and docking of 2′3′-cGAMP to all of them failed to return a thermodynamically viable pose (complex conformation). (M) Experimental scheme for N. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation or not with increasing doses of NU7441 (0, 0.2, 2, and 20 µM) followed by 2′3′-cGAMP incubation, release of bound 2′3′-cGAMP, and detection by ELISA (N) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in DNA-PK–specific IP performed as in M. n = 3 independent experiments. Statistical significance was calculated by two-tailed Student t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: . IP, immunoprecipitation.

Figure Legend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B. Whole-cell extracts (WCEs) from T98G cells were used as input for IPs using mock IgG and DNA-PKcs–specific antibodies prior to incubation with 2′3′-cGAMP and detection of bound 2′3′-cGAMP. (B) WB analysis of DNA-PKcs IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) Graph represents mean (±SEM; n = 3 independent experiment) 2′3′-cGAMP levels as measured in mock and DNA-PK–specific IP performed as in A. Statistical significance was calculated by two-tailed Student t test. (D) Silver staining was conducted on recombinant DNA-PKcs used in for immunoprecipitation experiments. Representative gel of three independent experiments. (E) WB analysis of TSA, conducted on WCE from THP-1 cells incubated with or without 2′3′-cGAMP or in presence or absence of NU7441. Immunoblot was performed using DNA-PKcs–, STING-, and HSP90-specific antibodies. Representative WB of three independent experiments. (F) Heatmap representation of the relative band intensities quantified from three independent experiments performed as in E. (G) Molecular modelling and docking study of 2′3′-cGAMP into DNA-PKcs. Human DNA-PKcs in ribbon representation with 2′3′-cGAMP docked in its catalytic site. (H) The docking conformation of 2′3′-cGAMP (in red spacefill representation) into the catalytic site of DNA-PKcs in the proximity of the catalytic residues (in ball and stick representation). (I) The docked conformation adopted by 2′3′-cGAMP onto the catalytic site of DNA-PKcs upon the MDSs. (J) The 2D molecular interactions diagram of 2′3′-cGAMP with the catalytic residues of DNA-PKcs. (K) Molecular modelling of ATM and ATR. DNA-PKcs superposed to the models of ATM and ATR (in red, blue, and yellow ribbon representations, respectively). (L) Close-up of the superposed active sites of ATM, ATR, and DNA-PKcs. Each of the three kinases has significant conformational differences and docking of 2′3′-cGAMP to all of them failed to return a thermodynamically viable pose (complex conformation). (M) Experimental scheme for N. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation or not with increasing doses of NU7441 (0, 0.2, 2, and 20 µM) followed by 2′3′-cGAMP incubation, release of bound 2′3′-cGAMP, and detection by ELISA (N) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in DNA-PK–specific IP performed as in M. n = 3 independent experiments. Statistical significance was calculated by two-tailed Student t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: . IP, immunoprecipitation.

Techniques Used: Incubation, Two Tailed Test, Silver Staining, Recombinant, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay

DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Figure Legend Snippet: DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Techniques Used: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

Image Search Results

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to immunoprecipitation (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: .

Article Snippet: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

Techniques: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B. Whole-cell extracts (WCEs) from T98G cells were used as input for IPs using mock IgG and DNA-PKcs–specific antibodies prior to incubation with 2′3′-cGAMP and detection of bound 2′3′-cGAMP. (B) WB analysis of DNA-PKcs IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) Graph represents mean (±SEM; n = 3 independent experiment) 2′3′-cGAMP levels as measured in mock and DNA-PK–specific IP performed as in A. Statistical significance was calculated by two-tailed Student t test. (D) Silver staining was conducted on recombinant DNA-PKcs used in for immunoprecipitation experiments. Representative gel of three independent experiments. (E) WB analysis of TSA, conducted on WCE from THP-1 cells incubated with or without 2′3′-cGAMP or in presence or absence of NU7441. Immunoblot was performed using DNA-PKcs–, STING-, and HSP90-specific antibodies. Representative WB of three independent experiments. (F) Heatmap representation of the relative band intensities quantified from three independent experiments performed as in E. (G) Molecular modelling and docking study of 2′3′-cGAMP into DNA-PKcs. Human DNA-PKcs in ribbon representation with 2′3′-cGAMP docked in its catalytic site. (H) The docking conformation of 2′3′-cGAMP (in red spacefill representation) into the catalytic site of DNA-PKcs in the proximity of the catalytic residues (in ball and stick representation). (I) The docked conformation adopted by 2′3′-cGAMP onto the catalytic site of DNA-PKcs upon the MDSs. (J) The 2D molecular interactions diagram of 2′3′-cGAMP with the catalytic residues of DNA-PKcs. (K) Molecular modelling of ATM and ATR. DNA-PKcs superposed to the models of ATM and ATR (in red, blue, and yellow ribbon representations, respectively). (L) Close-up of the superposed active sites of ATM, ATR, and DNA-PKcs. Each of the three kinases has significant conformational differences and docking of 2′3′-cGAMP to all of them failed to return a thermodynamically viable pose (complex conformation). (M) Experimental scheme for N. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation or not with increasing doses of NU7441 (0, 0.2, 2, and 20 µM) followed by 2′3′-cGAMP incubation, release of bound 2′3′-cGAMP, and detection by ELISA (N) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in DNA-PK–specific IP performed as in M. n = 3 independent experiments. Statistical significance was calculated by two-tailed Student t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to . Source data are available for this figure: . IP, immunoprecipitation.

Article Snippet: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

Techniques: Incubation, Two Tailed Test, Silver Staining, Recombinant, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . IP, immunoprecipitation.

Article Snippet: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

BRSK2 expression correlates with clinical outcomes in patients with breast cancer. ( A ) Boxplots show elevated log2 expression of BRSK2 mRNA in breast cancer patients from the TCGA-BRCA whole cohort (n = 1092) compared to normal adjacent breast tissue (n = 112). *** p < 0.001 (tumor vs. normal). ( B ) Boxplots show elevated BRSK2 transcript levels in metastatic patients (n = 7) from the TCGA-BRCA cohort compared to normal adjacent breast tissues (n = 112). ( C ) Boxplots of high expression of BRSK2 mRNA of tumors of different American Joint Committee on Cancer (AJCC) stages for TCGA-BRCA cohort. ( D ) Boxplots of high BRSK2 expression score by immunohistochemistry (IHC) determined subtype in the TCGA-BRCA cohort. All boxes are presented as medians with interquartile range. Mann–Whitney U and Kruskal–Wallis H tests were used for the analysis. For some figures, significant p -values are shown. * p < 0.05 was considered significant. ( E ) Kaplan–Meier plots with log-rank p -values for disease-specific survival (DSS) in the TCGA-BRCA cohort, stratified by BRSK2 mRNA expression. Adverse DSS with higher BRSK2 transcripts was found in the TCGA-BRCA cohort, and the log − rank test was used for the analysis. The median split patients and significant p -values are shown ( p = 0.005). ( F ) BRSK2 transcripts were analyzed by quantitative PCR (qPCR) in patients with primary tumors. Primary tumors (never-metastatic, n = 10) and age-matched primary tumors from patients with a history of metastasis (n = 10) were used for qPCR analysis of BRSK2 and GAPGH as a housekeeping control gene. Normalized BRSK2 gene levels to GAPDH (2^ −delta CTx10,000) are shown. The student’s t-test, ** p < 0.01, is significant in experimental triplicate.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 expression correlates with clinical outcomes in patients with breast cancer. ( A ) Boxplots show elevated log2 expression of BRSK2 mRNA in breast cancer patients from the TCGA-BRCA whole cohort (n = 1092) compared to normal adjacent breast tissue (n = 112). *** p < 0.001 (tumor vs. normal). ( B ) Boxplots show elevated BRSK2 transcript levels in metastatic patients (n = 7) from the TCGA-BRCA cohort compared to normal adjacent breast tissues (n = 112). ( C ) Boxplots of high expression of BRSK2 mRNA of tumors of different American Joint Committee on Cancer (AJCC) stages for TCGA-BRCA cohort. ( D ) Boxplots of high BRSK2 expression score by immunohistochemistry (IHC) determined subtype in the TCGA-BRCA cohort. All boxes are presented as medians with interquartile range. Mann–Whitney U and Kruskal–Wallis H tests were used for the analysis. For some figures, significant p -values are shown. * p < 0.05 was considered significant. ( E ) Kaplan–Meier plots with log-rank p -values for disease-specific survival (DSS) in the TCGA-BRCA cohort, stratified by BRSK2 mRNA expression. Adverse DSS with higher BRSK2 transcripts was found in the TCGA-BRCA cohort, and the log − rank test was used for the analysis. The median split patients and significant p -values are shown ( p = 0.005). ( F ) BRSK2 transcripts were analyzed by quantitative PCR (qPCR) in patients with primary tumors. Primary tumors (never-metastatic, n = 10) and age-matched primary tumors from patients with a history of metastasis (n = 10) were used for qPCR analysis of BRSK2 and GAPGH as a housekeeping control gene. Normalized BRSK2 gene levels to GAPDH (2^ −delta CTx10,000) are shown. The student’s t-test, ** p < 0.01, is significant in experimental triplicate.

Article Snippet: For endogenous BRSK2 immunoprecipitation, 500 μg total proteins from BT-549 cells treated with or without a nutrient-free medium (6 h) were immunoprecipitated with antibodies against anti-BRSK2 antibodies (#11589-1-AP, Proteintech, USA) or control normal IgG.

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Control

BRSK2 is elevated in human breast cancer cells vs. benign tumor cells. ( A ), Total cell lysates from normal human breast epithelial cell lines (MCF10A), an array of human breast cancer ER+ cell lines, and TNBC and metastatic TNBC cell lines were used for western blot analysis with the antibodies against BRSK2 ( A ). Since BRSK2 is primarily expressed in the brain, a mouse whole-brain protein extract was used as a positive control for BRSK2 protein expression. GAPDH was used to show equal loading and transfer. N = 3, representative blots were shown. The BRSK2 protein band (78–90 kDa) in the western blot. ( B ) Total RNA was isolated separately from individual cells and treated with DNase. cDNA was prepared from total RNA and used for SYBR-Green qPCR analysis for BRSK2 transcript expression. GAPDH mRNA expression was used to normalize BRSK2 transcript levels. Normalized transcript expression levels were calculated using the Delta CT method (n = 3); data are means ± s.e. One-way ANOVA (*** p < 0.001), Student’s t-test, and *** p < 0.001 (metastatic TNBC cell vs. parental TNBC cell). Note : Normalized BRSK2 mRNA levels are significantly elevated (*** p < 0.001, cancer cells vs. MCF10A).

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 is elevated in human breast cancer cells vs. benign tumor cells. ( A ), Total cell lysates from normal human breast epithelial cell lines (MCF10A), an array of human breast cancer ER+ cell lines, and TNBC and metastatic TNBC cell lines were used for western blot analysis with the antibodies against BRSK2 ( A ). Since BRSK2 is primarily expressed in the brain, a mouse whole-brain protein extract was used as a positive control for BRSK2 protein expression. GAPDH was used to show equal loading and transfer. N = 3, representative blots were shown. The BRSK2 protein band (78–90 kDa) in the western blot. ( B ) Total RNA was isolated separately from individual cells and treated with DNase. cDNA was prepared from total RNA and used for SYBR-Green qPCR analysis for BRSK2 transcript expression. GAPDH mRNA expression was used to normalize BRSK2 transcript levels. Normalized transcript expression levels were calculated using the Delta CT method (n = 3); data are means ± s.e. One-way ANOVA (*** p < 0.001), Student’s t-test, and *** p < 0.001 (metastatic TNBC cell vs. parental TNBC cell). Note : Normalized BRSK2 mRNA levels are significantly elevated (*** p < 0.001, cancer cells vs. MCF10A).

Techniques: Western Blot, Positive Control, Expressing, Isolation, SYBR Green Assay

$BRSK2 ectopic expression in breast cancer cells is associated with tumor cell survival pathways linked to AKT and activation of master transcription factors STAT3 and NF-κB. BT-474 ( A ), MDA-MB-231 ( B ), and MDA-MB-231/BR ( C ) cells were transfected with the empty vector, BRSK2, or the BRSK1 plasmids for 48 h, and total protein lysates were prepared for western blot analysis with the indicated antibodies. For some experiments, 3D cell cultures were established 24 h after plasmid transfection. ( D ) MDA-MB-231/BR cells, as shown, were grown for an additional 48 h in a low-attachment six-well plate to form tumorospheres (see Materials and Methods). Total protein lysates were prepared for western blot analysis, as indicated, including phospho-AKT (Ser473), phosphor-STAT3 (Ser727 and Tyr705), and phospho-NF-κB (Ser536) antibodies. CHC and β-actin antibodies were used for equal loading and transfer. Anti-FLAG antibodies were used for FLAG-tagged BRSK2 protein expression. n = 3, representative blots are shown. Duplicate BT-474 ( A ) and MDA-MB-231 ( B ) cell cultures were used to isolate nuclear and cytosolic fractions, free of nuclei, using established protocols. ( E and F ) As indicated, equal amounts of nuclear and cytosolic proteins were used for Western blotting, using specific antibodies against the target proteins. Translocation of p65-NF-κB or active STAT3(Y705) into the nucleus in response to ectopic BRSK2 or BRSK1 expression vs. empty vector was observed in both BT474 ( E ) and MDA-MB-231 ( F ) cell models. α-tubulin and total ERK1/2 antibodies, as well as histone H3 and Lamin B1 antibodies, were used to identify cytosolic and nuclear protein markers, respectively, free of nuclei. n = 3, representative blots were shown. Scanned blots and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ).$

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 ectopic expression in breast cancer cells is associated with tumor cell survival pathways linked to AKT and activation of master transcription factors STAT3 and NF-κB. BT-474 ( A ), MDA-MB-231 ( B ), and MDA-MB-231/BR ( C ) cells were transfected with the empty vector, BRSK2, or the BRSK1 plasmids for 48 h, and total protein lysates were prepared for western blot analysis with the indicated antibodies. For some experiments, 3D cell cultures were established 24 h after plasmid transfection. ( D ) MDA-MB-231/BR cells, as shown, were grown for an additional 48 h in a low-attachment six-well plate to form tumorospheres (see Materials and Methods). Total protein lysates were prepared for western blot analysis, as indicated, including phospho-AKT (Ser473), phosphor-STAT3 (Ser727 and Tyr705), and phospho-NF-κB (Ser536) antibodies. CHC and β-actin antibodies were used for equal loading and transfer. Anti-FLAG antibodies were used for FLAG-tagged BRSK2 protein expression. n = 3, representative blots are shown. Duplicate BT-474 ( A ) and MDA-MB-231 ( B ) cell cultures were used to isolate nuclear and cytosolic fractions, free of nuclei, using established protocols. ( E and F ) As indicated, equal amounts of nuclear and cytosolic proteins were used for Western blotting, using specific antibodies against the target proteins. Translocation of p65-NF-κB or active STAT3(Y705) into the nucleus in response to ectopic BRSK2 or BRSK1 expression vs. empty vector was observed in both BT474 ( E ) and MDA-MB-231 ( F ) cell models. α-tubulin and total ERK1/2 antibodies, as well as histone H3 and Lamin B1 antibodies, were used to identify cytosolic and nuclear protein markers, respectively, free of nuclei. n = 3, representative blots were shown. Scanned blots and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ).

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Translocation Assay, Control, Labeling

Ectopic BRSK2 expression in breast cancer cells is associated with elevated autophagy in response to nutrient deprivation stress. ( A ) BT-474 cells were transfected with empty vector or BRSK2 overexpression plasmids for 48 h and subjected to exposure with various conditions with or without deprived energy sources medium (glucose, glutamine, or nutrients) for 0 h to 6 h, as indicated. Equal amounts of protein were used for Western blot analysis with autophagy-related protein antibodies. The total cell lysate was also analyzed by western blot, probed with an anti-FLAG antibody to reconfirm BRSK2 protein overexpression, and an anti-β-Actin antibody to confirm equal loading. ( B ) LC3–II band intensities (normalized to β-actin) were quantified by densitometry, and the measurements were used to calculate autophagy for each treatment separately (n = 4 independent experiments). BRSK2 overexpression increased autophagy in 6 h in response to nutrient-deprived stress. ** p < 0.01 and *** p < 0.001 (BRSK2 OE vs. corresponding empty vector control). ( C – E ) MDA-MB-231 cells were transiently transfected with either an empty vector or a green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h, followed by an additional 6 h of nutrient starvation. ( C ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3) and ( D ) quantification (puncta/cell) confirmed that BRSK2-mediated autophagosomes were increased within 6 h of nutrient starvation in MDA-MB-231 cells. During fluorescent microscopy, exposure time and weighting for GFP fluorescence were kept consistent between samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta staining was counted from at least n = 20 cells/sample to quantify autophagic puncta formation. Bar plots display data from autophagic puncta quantification assays, with means ± s.e. One-way ANOVA and post-hoc tests for pairwise comparisons are shown; * p < 0.05, *** p < 0.001. ( E ) Similar experiments were performed in MDA-MB-231 cells exposed to ± nutrient-free medium for 6 h only, and total protein lysates were used for western blot analysis using the indicated antibodies. An anti-β-Actin antibody was used to ensure equal loading and transfer. For all western blotting experiments (n = 3), representative blots are shown. Fold LC3-II band and other autophagic protein band intensities normalized to β-Actin vs. control vector-transfected cells were labeled (ImageJ).

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: Ectopic BRSK2 expression in breast cancer cells is associated with elevated autophagy in response to nutrient deprivation stress. ( A ) BT-474 cells were transfected with empty vector or BRSK2 overexpression plasmids for 48 h and subjected to exposure with various conditions with or without deprived energy sources medium (glucose, glutamine, or nutrients) for 0 h to 6 h, as indicated. Equal amounts of protein were used for Western blot analysis with autophagy-related protein antibodies. The total cell lysate was also analyzed by western blot, probed with an anti-FLAG antibody to reconfirm BRSK2 protein overexpression, and an anti-β-Actin antibody to confirm equal loading. ( B ) LC3–II band intensities (normalized to β-actin) were quantified by densitometry, and the measurements were used to calculate autophagy for each treatment separately (n = 4 independent experiments). BRSK2 overexpression increased autophagy in 6 h in response to nutrient-deprived stress. ** p < 0.01 and *** p < 0.001 (BRSK2 OE vs. corresponding empty vector control). ( C – E ) MDA-MB-231 cells were transiently transfected with either an empty vector or a green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h, followed by an additional 6 h of nutrient starvation. ( C ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3) and ( D ) quantification (puncta/cell) confirmed that BRSK2-mediated autophagosomes were increased within 6 h of nutrient starvation in MDA-MB-231 cells. During fluorescent microscopy, exposure time and weighting for GFP fluorescence were kept consistent between samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta staining was counted from at least n = 20 cells/sample to quantify autophagic puncta formation. Bar plots display data from autophagic puncta quantification assays, with means ± s.e. One-way ANOVA and post-hoc tests for pairwise comparisons are shown; * p < 0.05, *** p < 0.001. ( E ) Similar experiments were performed in MDA-MB-231 cells exposed to ± nutrient-free medium for 6 h only, and total protein lysates were used for western blot analysis using the indicated antibodies. An anti-β-Actin antibody was used to ensure equal loading and transfer. For all western blotting experiments (n = 3), representative blots are shown. Fold LC3-II band and other autophagic protein band intensities normalized to β-Actin vs. control vector-transfected cells were labeled (ImageJ).

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Western Blot, Control, Staining, Microscopy, Fluorescence, Labeling

BRSK2 overexpression enhances autophagy flux in breast cancer cells in response to nutrient deprivation stress. ( A ) BT-474 and ( D ) MDA-MB-231 cells were transfected with empty vector or BRSK2 overexpression plasmids for 48 h and subjected to nutrient-null medium ± BafA1 (100 nM) exposure for four h. Equal amounts of protein extracts from each sample were used for western blot analysis with the indicated antibodies—LC3, FLAG, and GAPDH—to ensure equal loading and transfer. Short and long-exposure FLAG-BRSK2 western blots were shown. n = 3, representative blots were shown. Fold LC3-II band intensities normalized to GAPDH vs. control vector-transfected cells were labeled (ImageJ). ( B, C, and E, F) GFP-LC3 puncta staining and quantification. BT-474 ( B, C ) and MDA-MB-231 cells ( E, F ) were transiently transfected with either empty vector, BRSK2, or green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h followed by an additional 4 h of nutrient starvation ± BafA1 (100 nM). ( B, E ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3, red arrows) and ( C, F ) quantification (puncta/cell) confirmed that BRSK2-mediated autophagosomes were increased within 4 h of nutrient starvation in MDA-MB-231 cells. During fluorescent microscopy, the exposure time and GFP fluorescence weighting were kept consistent across all samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta were counted in at least n = 20 cells/sample to quantify autophagic changes. Scatter plots of data for autophagic puncta quantification assays are shown as means ± s.e., with one-way ANOVA and post hoc tests for pairwise comparisons; *** p < 0.001.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 overexpression enhances autophagy flux in breast cancer cells in response to nutrient deprivation stress. ( A ) BT-474 and ( D ) MDA-MB-231 cells were transfected with empty vector or BRSK2 overexpression plasmids for 48 h and subjected to nutrient-null medium ± BafA1 (100 nM) exposure for four h. Equal amounts of protein extracts from each sample were used for western blot analysis with the indicated antibodies—LC3, FLAG, and GAPDH—to ensure equal loading and transfer. Short and long-exposure FLAG-BRSK2 western blots were shown. n = 3, representative blots were shown. Fold LC3-II band intensities normalized to GAPDH vs. control vector-transfected cells were labeled (ImageJ). ( B, C, and E, F) GFP-LC3 puncta staining and quantification. BT-474 ( B, C ) and MDA-MB-231 cells ( E, F ) were transiently transfected with either empty vector, BRSK2, or green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h followed by an additional 4 h of nutrient starvation ± BafA1 (100 nM). ( B, E ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3, red arrows) and ( C, F ) quantification (puncta/cell) confirmed that BRSK2-mediated autophagosomes were increased within 4 h of nutrient starvation in MDA-MB-231 cells. During fluorescent microscopy, the exposure time and GFP fluorescence weighting were kept consistent across all samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta were counted in at least n = 20 cells/sample to quantify autophagic changes. Scatter plots of data for autophagic puncta quantification assays are shown as means ± s.e., with one-way ANOVA and post hoc tests for pairwise comparisons; *** p < 0.001.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control, Labeling, Staining, Microscopy, Fluorescence

Knockdown of BRSK2 suppresses AKT, STAT3, and NF-κB signaling, as well as basal autophagy and autophagy induced by nutrient deprivation stress, in breast cancer cells. ( A ) MDA-MB-231 cells transfected with siControl and validated siRNA targeted to BRSK2 were used for total protein lysate isolation, followed by western blot analysis with antibodies against the indicated proteins, including autophagy-related proteins p62/SQSTM1, ATG3, Beclin-1, active AKT (Ser473), STAT3 (Ser727 and Tyr705), phospho-ERK1/2, and NF-κB (Ser536). An anti-β-actin antibody was used to ensure equal loading and transfer. ( B ) BRSK2 is knocked down in MDA-MB-231 cells using siRNA targeted to BRSK2, compared to siControl. Knockdown of BRSK2 levels was rescued by ectopic overexpression of BRSK2. Equal amounts of total protein were used for Western blotting with antibodies against LC3, BRSK2, and FLAG-BRSK2. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. ( C ) BRSK2 protein was knocked down in BT-474 cells using two different siRNAs (siBRSK2 #1 and siBRSK2 #2) compared to siControl. Cells were exposed to ± nutrient-deprived medium for 6 h and subjected to total protein lysate isolation followed by western blotting. ( D ) BRSK2-knocked-down BT-474 cells with siRNA targeted to BRSK2 (siBRSK2 #1) ± rescued by overexpression of BRSK2 were used for total lysate isolation, followed by western blotting with antibodies against the indicated proteins, including BRSK2 and phospho-AKT. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. All western blotting experiments (n = 3) and representative blots are shown. Blots were scanned, and fold intensities normalized to respective loading control (β Actin) vs. vector control were labeled (ImageJ).

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: Knockdown of BRSK2 suppresses AKT, STAT3, and NF-κB signaling, as well as basal autophagy and autophagy induced by nutrient deprivation stress, in breast cancer cells. ( A ) MDA-MB-231 cells transfected with siControl and validated siRNA targeted to BRSK2 were used for total protein lysate isolation, followed by western blot analysis with antibodies against the indicated proteins, including autophagy-related proteins p62/SQSTM1, ATG3, Beclin-1, active AKT (Ser473), STAT3 (Ser727 and Tyr705), phospho-ERK1/2, and NF-κB (Ser536). An anti-β-actin antibody was used to ensure equal loading and transfer. ( B ) BRSK2 is knocked down in MDA-MB-231 cells using siRNA targeted to BRSK2, compared to siControl. Knockdown of BRSK2 levels was rescued by ectopic overexpression of BRSK2. Equal amounts of total protein were used for Western blotting with antibodies against LC3, BRSK2, and FLAG-BRSK2. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. ( C ) BRSK2 protein was knocked down in BT-474 cells using two different siRNAs (siBRSK2 #1 and siBRSK2 #2) compared to siControl. Cells were exposed to ± nutrient-deprived medium for 6 h and subjected to total protein lysate isolation followed by western blotting. ( D ) BRSK2-knocked-down BT-474 cells with siRNA targeted to BRSK2 (siBRSK2 #1) ± rescued by overexpression of BRSK2 were used for total lysate isolation, followed by western blotting with antibodies against the indicated proteins, including BRSK2 and phospho-AKT. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. All western blotting experiments (n = 3) and representative blots are shown. Blots were scanned, and fold intensities normalized to respective loading control (β Actin) vs. vector control were labeled (ImageJ).

Techniques: Knockdown, Transfection, Isolation, Western Blot, Over Expression, Control, Plasmid Preparation, Labeling

BRSK2 downregulation reduces autophagy flux and autophagosome formation in breast cancer cells in response to nutrient deprivation stress and bafilomycin A1. ( A ) BT-549, ( B ) MDA-MB-231, and ( C ) BT-474 cells were transfected with siControl or siBRSK2 #1, or stBRSK2 #2 for 48 h and subjected to nutrient-null medium ± BafA1 (100 nM) exposure for 4 h. Equal amounts of protein extracts from each sample were used for western blot analysis with the indicated antibodies, including LC3, BRSK2, and GAPDH, as equal loading and transfer. N = 3, representative blots were shown. Fold LC3-II band intensities normalized to GAPDH vs. siControl transfected cells were labeled (ImageJ). ( D and E ) GFP-LC3 puncta staining and quantification. BT-549 cells were transfected with siControl, siBRSK2 #1, or green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h followed by an additional 4 h of nutrient starvation ± BafA1 (100 nM). ( D ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3, red arrows) and ( E ) quantification (puncta/cell) confirmed that siBRSK2-mediated autophagosomes were decreased within 4 h of nutrient starvation and BafA1 exposure in BT-549 cells. During fluorescent microscopy, the exposure time and weighting for GFP fluorescence were kept consistent across all samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta staining was counted from at least n = 20 cells/sample to quantify autophagic puncta formation. Autophagic puncta quantification assays are means ± s.e., one-way ANOVA, and post-hoc test for pair-wise comparison, * p < 0.05 or *** p < 0.001 vs. siControl.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 downregulation reduces autophagy flux and autophagosome formation in breast cancer cells in response to nutrient deprivation stress and bafilomycin A1. ( A ) BT-549, ( B ) MDA-MB-231, and ( C ) BT-474 cells were transfected with siControl or siBRSK2 #1, or stBRSK2 #2 for 48 h and subjected to nutrient-null medium ± BafA1 (100 nM) exposure for 4 h. Equal amounts of protein extracts from each sample were used for western blot analysis with the indicated antibodies, including LC3, BRSK2, and GAPDH, as equal loading and transfer. N = 3, representative blots were shown. Fold LC3-II band intensities normalized to GAPDH vs. siControl transfected cells were labeled (ImageJ). ( D and E ) GFP-LC3 puncta staining and quantification. BT-549 cells were transfected with siControl, siBRSK2 #1, or green fluorescent protein (GFP)–tagged LC3 plasmid for 48 h followed by an additional 4 h of nutrient starvation ± BafA1 (100 nM). ( D ) Microscopic images of autophagosome formation (puncta staining of GFP-LC3, red arrows) and ( E ) quantification (puncta/cell) confirmed that siBRSK2-mediated autophagosomes were decreased within 4 h of nutrient starvation and BafA1 exposure in BT-549 cells. During fluorescent microscopy, the exposure time and weighting for GFP fluorescence were kept consistent across all samples. Representative images (n = 10) were shown on a 25–27 µm scale bar. In addition, GFP-LC3 puncta staining was counted from at least n = 20 cells/sample to quantify autophagic puncta formation. Autophagic puncta quantification assays are means ± s.e., one-way ANOVA, and post-hoc test for pair-wise comparison, * p < 0.05 or *** p < 0.001 vs. siControl.

Techniques: Transfection, Western Blot, Labeling, Staining, Plasmid Preparation, Microscopy, Fluorescence, Comparison

Suppression of BRSK2 expression or inhibition by GW296115 reduces breast cancer cell growth and autophagy, thereby enhancing their apoptosis induced by nutrient deprivation stress. BT-474 ( A ) and MDA-MB-231 ( B ) cells were transfected with siRNA (Dharmacon, #1) targeted to BRSK2 or control siRNA for 48 h, and cells were exposed to ± nutrient-deprived medium for another 6 h, followed by total protein lysate isolation for western blotting analysis. BRSK2 downregulation efficiencies were validated by western blotting with anti-BRSK2 antibodies. Autophagy marker protein antibodies (anti-LC3) and apoptotic marker protein antibodies (anti-cleaved PARP, anti-cleaved caspase-7, and anti-cleaved caspase-9) were used, along with β-actin for equal loading. Proteins from duplicate cultures were resolved by SDS-PAGE, and cytochrome c released from mitochondria into the cytosol was assessed by immunoblotting using antibodies against cytochrome c (Cyc-c) and β-tubulin to ensure equal loading and transfer. Similar results were obtained in three additional experiments. All the apoptosis Western blots were also confirmed with siBRSK2 #2 (Qiagen, not shown). ( C ) Pharmacological inhibition of BRSK2, using GW296115 (2–5 µM) versus vehicle, decreased LC3-I/II protein levels but enhanced cleaved caspase. 7 and PARP proteins, as determined by western blotting in MDA-MB-231 cells. β-Actin was used as a loading control and for equal transfer. N = 3, representative blots were shown—similar data obtained in other breast cancer cells. All the blots were scanned, and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ). ( D ) Inhibition of BRSK2 kinase via GW296115 (2 µM) for 24 h reduced MDA-MB-231 cell growth, n = 5, Student’s t-test, *** p < 0.001( GW296115 vs. vehicle control). ( E ) GW296115 (2–3 µM) vs. vehicle treatment for 72 h significantly reduced 3D Matrigel tumor cell invasiveness (invadopodia) in MDA-MB-231 cells. Representative light microscopic images (n = 10), scale bar 200 µm, quantification of invadopodia/spheroid, n = 10, ANOVA and post-hoc t-test, *** p < 0.001 vs. vehicle.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: Suppression of BRSK2 expression or inhibition by GW296115 reduces breast cancer cell growth and autophagy, thereby enhancing their apoptosis induced by nutrient deprivation stress. BT-474 ( A ) and MDA-MB-231 ( B ) cells were transfected with siRNA (Dharmacon, #1) targeted to BRSK2 or control siRNA for 48 h, and cells were exposed to ± nutrient-deprived medium for another 6 h, followed by total protein lysate isolation for western blotting analysis. BRSK2 downregulation efficiencies were validated by western blotting with anti-BRSK2 antibodies. Autophagy marker protein antibodies (anti-LC3) and apoptotic marker protein antibodies (anti-cleaved PARP, anti-cleaved caspase-7, and anti-cleaved caspase-9) were used, along with β-actin for equal loading. Proteins from duplicate cultures were resolved by SDS-PAGE, and cytochrome c released from mitochondria into the cytosol was assessed by immunoblotting using antibodies against cytochrome c (Cyc-c) and β-tubulin to ensure equal loading and transfer. Similar results were obtained in three additional experiments. All the apoptosis Western blots were also confirmed with siBRSK2 #2 (Qiagen, not shown). ( C ) Pharmacological inhibition of BRSK2, using GW296115 (2–5 µM) versus vehicle, decreased LC3-I/II protein levels but enhanced cleaved caspase. 7 and PARP proteins, as determined by western blotting in MDA-MB-231 cells. β-Actin was used as a loading control and for equal transfer. N = 3, representative blots were shown—similar data obtained in other breast cancer cells. All the blots were scanned, and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ). ( D ) Inhibition of BRSK2 kinase via GW296115 (2 µM) for 24 h reduced MDA-MB-231 cell growth, n = 5, Student’s t-test, *** p < 0.001( GW296115 vs. vehicle control). ( E ) GW296115 (2–3 µM) vs. vehicle treatment for 72 h significantly reduced 3D Matrigel tumor cell invasiveness (invadopodia) in MDA-MB-231 cells. Representative light microscopic images (n = 10), scale bar 200 µm, quantification of invadopodia/spheroid, n = 10, ANOVA and post-hoc t-test, *** p < 0.001 vs. vehicle.

Techniques: Expressing, Inhibition, Transfection, Control, Isolation, Western Blot, Marker, SDS Page, Plasmid Preparation, Labeling

BRSK2 is involved in nutrient deprivation stress-mediated autophagic vacuole formation. ( A ) The endogenous BRSK2 gene was knocked down by CRISPR-Cas9 vs. CRISPR control in MDA-MB-231 cells, and a stable BRSK2 knockout clone was selected and verified by western blotting with anti-BRSK2 antibodies. As expected, knockout cells have markedly reduced LC3-II levels, P-ERK1/2, P-AKT, and P-p65-NF-kB protein levels. GAPDH was used for equal loading and transfer in Western blotting. Experiments were repeated three times, and representative blots are shown. Blots were scanned, and protein bands were quantified (ImageJ). ( B ) BRSK2 knockout MDA-MB-231 cells vs. control cells were exposed to ± nutrient-free medium for 6 h and subjected to TEM analysis, n = 10, representative image, scale bar 200 µm, and red arrows indicate autophagosome vacuoles. ( C ) Duplicate cells from ( B ) were transfected with GFP-LC3 and treated with or without nutrient-free medium for 6 h, followed by GFP immunofluorescence staining. Representative inverted fluorescence microscopic images, n = 10, scale bar 100 µm, red arrows on blowout images indicate autophagy puncta GFP-LC3 staining (n = 20 cells). ANOVA and post-hoc t-test, *** p < 0.001 vs. vehicle. ( D ) BRSK2 overexpression in knockout cells enhances autophagy in response to nutrient deprivation. BRSK2 was overexpressed in BRSK2 knockout MDA-MB-231 cells, followed by GFP-LC3 overexpression. Cells were then exposed to ± nutrient-free medium for 6 h and followed by GFP immunofluorescence imaging. Representative inverted fluorescence microscopic images were shown, n = 10, scale bar 100 µm, red arrows on blowout image, indicating autophagy GFP-LC3 puncta staining (n = 20 cells). Box plots are the number of dots/cells from at least n = 20 counted, Student’s t-test, *** p < 0.001 (nutrient-free vs. nutrient plus medium). KD, knockdown.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 is involved in nutrient deprivation stress-mediated autophagic vacuole formation. ( A ) The endogenous BRSK2 gene was knocked down by CRISPR-Cas9 vs. CRISPR control in MDA-MB-231 cells, and a stable BRSK2 knockout clone was selected and verified by western blotting with anti-BRSK2 antibodies. As expected, knockout cells have markedly reduced LC3-II levels, P-ERK1/2, P-AKT, and P-p65-NF-kB protein levels. GAPDH was used for equal loading and transfer in Western blotting. Experiments were repeated three times, and representative blots are shown. Blots were scanned, and protein bands were quantified (ImageJ). ( B ) BRSK2 knockout MDA-MB-231 cells vs. control cells were exposed to ± nutrient-free medium for 6 h and subjected to TEM analysis, n = 10, representative image, scale bar 200 µm, and red arrows indicate autophagosome vacuoles. ( C ) Duplicate cells from ( B ) were transfected with GFP-LC3 and treated with or without nutrient-free medium for 6 h, followed by GFP immunofluorescence staining. Representative inverted fluorescence microscopic images, n = 10, scale bar 100 µm, red arrows on blowout images indicate autophagy puncta GFP-LC3 staining (n = 20 cells). ANOVA and post-hoc t-test, *** p < 0.001 vs. vehicle. ( D ) BRSK2 overexpression in knockout cells enhances autophagy in response to nutrient deprivation. BRSK2 was overexpressed in BRSK2 knockout MDA-MB-231 cells, followed by GFP-LC3 overexpression. Cells were then exposed to ± nutrient-free medium for 6 h and followed by GFP immunofluorescence imaging. Representative inverted fluorescence microscopic images were shown, n = 10, scale bar 100 µm, red arrows on blowout image, indicating autophagy GFP-LC3 puncta staining (n = 20 cells). Box plots are the number of dots/cells from at least n = 20 counted, Student’s t-test, *** p < 0.001 (nutrient-free vs. nutrient plus medium). KD, knockdown.

Techniques: CRISPR, Control, Knock-Out, Western Blot, Transfection, Immunofluorescence, Staining, Fluorescence, Over Expression, Imaging, Knockdown

BRSK2 is involved in the formation of the Vps34-class III PI3K-Beclin-1-ATG14 autophagy signaling complex. ( A and B ) BRSK2 was ectopically overexpressed in MDA-MB-231 BRSK2 knockout cells. Vector-transfected control cells and BRSK2-overexpressed cells were exposed to a nutrient-free medium with or without nutrients for 6 h. As indicated, equal amounts of total cell lysate protein were immunoprecipitated with control IgG and anti-FLAG antibodies. Inputs ( A ) and immunoprecipitated BRSK2-protein complexes with anti-FLAG antibodies ( B ) were resolved in SDS-PAGE for western blotting with the indicated protein antibodies. Anti-FLAG antibodies were used to detect the FLAG-tagged BRSK2 protein. ( C – E ) BRSK2 interacts with ATG14 in vitro. ( C ) FLAG-BRSK2 and GFP-ATG14 were ectopically overexpressed in MDA-MB-231 cells with an empty vector. Equal proteins from cell extracts were used for western blot analysis (inputs) to demonstrate FLAG-BRSK2 (top panel) and GFP-ATG14 (bottom panel) protein expression in these cells. ( D ) 500 µg total proteins from each sample were used in FLAG-BRSK2 IP (top panel) and GFP-ATG14 GFP-beads pulldown (bottom panel) experiments. Eluted proteins from extensively washed FLAG-IP Protein G beads and pulldown GFP beads were used for western blots and subjected to in vitro binding and western blot analysis, as indicated ( E ). FLAG-IP eluted proteins (vector and BRSK2) were separately incubated with the GFP-beads eluted proteins (Vector and ATG14) for 30 min at room temperature. GFP-bead pulldown captured GFP-tagged ATG14 and bound BRSK2 and was analyzed by Western blot with the indicated antibodies. ( F ) With or without nutrient-starved (6 h) BT-549 cells, equal protein amounts were incubated with anti-BRSK2 or control IgG antibodies to immunoprecipitate endogenous BRSK2 protein complexes. Input proteins and anti-BRSK2 immunoprecipitated protein complexes were resolved in SDS-PAGE for western blotting with the indicated protein antibodies. A whole-mouse-brain protein extract (1/10th of the input) was used as a positive control for BRSK2. GAPDH was used to load and transfer the input samples equally. Western blots (n = 3) and representative blots are shown. ( G ) We propose that BRSK2-mediated autophagy and tumor cell survival signaling in response to nutrient-rich or nutrient-deprived stress involve the class III PI3K-Vps34-Beclin-1-ATG14 pathway in breast cancer cell models.

Journal: Scientific Reports

Article Title: BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway

doi: 10.1038/s41598-025-24354-4

Figure Lengend Snippet: BRSK2 is involved in the formation of the Vps34-class III PI3K-Beclin-1-ATG14 autophagy signaling complex. ( A and B ) BRSK2 was ectopically overexpressed in MDA-MB-231 BRSK2 knockout cells. Vector-transfected control cells and BRSK2-overexpressed cells were exposed to a nutrient-free medium with or without nutrients for 6 h. As indicated, equal amounts of total cell lysate protein were immunoprecipitated with control IgG and anti-FLAG antibodies. Inputs ( A ) and immunoprecipitated BRSK2-protein complexes with anti-FLAG antibodies ( B ) were resolved in SDS-PAGE for western blotting with the indicated protein antibodies. Anti-FLAG antibodies were used to detect the FLAG-tagged BRSK2 protein. ( C – E ) BRSK2 interacts with ATG14 in vitro. ( C ) FLAG-BRSK2 and GFP-ATG14 were ectopically overexpressed in MDA-MB-231 cells with an empty vector. Equal proteins from cell extracts were used for western blot analysis (inputs) to demonstrate FLAG-BRSK2 (top panel) and GFP-ATG14 (bottom panel) protein expression in these cells. ( D ) 500 µg total proteins from each sample were used in FLAG-BRSK2 IP (top panel) and GFP-ATG14 GFP-beads pulldown (bottom panel) experiments. Eluted proteins from extensively washed FLAG-IP Protein G beads and pulldown GFP beads were used for western blots and subjected to in vitro binding and western blot analysis, as indicated ( E ). FLAG-IP eluted proteins (vector and BRSK2) were separately incubated with the GFP-beads eluted proteins (Vector and ATG14) for 30 min at room temperature. GFP-bead pulldown captured GFP-tagged ATG14 and bound BRSK2 and was analyzed by Western blot with the indicated antibodies. ( F ) With or without nutrient-starved (6 h) BT-549 cells, equal protein amounts were incubated with anti-BRSK2 or control IgG antibodies to immunoprecipitate endogenous BRSK2 protein complexes. Input proteins and anti-BRSK2 immunoprecipitated protein complexes were resolved in SDS-PAGE for western blotting with the indicated protein antibodies. A whole-mouse-brain protein extract (1/10th of the input) was used as a positive control for BRSK2. GAPDH was used to load and transfer the input samples equally. Western blots (n = 3) and representative blots are shown. ( G ) We propose that BRSK2-mediated autophagy and tumor cell survival signaling in response to nutrient-rich or nutrient-deprived stress involve the class III PI3K-Vps34-Beclin-1-ATG14 pathway in breast cancer cell models.

Techniques: Knock-Out, Plasmid Preparation, Transfection, Control, Immunoprecipitation, SDS Page, Western Blot, In Vitro, Expressing, Binding Assay, Incubation, Positive Control

( A ) Differentially expressed proteins in BAG3 –/– CFs. ( B and C ) STRING analysis of protein-protein interactions reveals 2 clusters of cell cycle– and ECM-associated protein networks. ( D ) Gene set enrichment analysis of TGF-β signaling pathway. ( E ) Fold change values for the TGF-β ligands and receptors identified by mass spectrometry. * P < 0.05 by 2-tailed Student’s t test with permutation-based FDR correction less than 0.05. n = 3 independent differentiations in mass spectrometry data.

Journal: The Journal of Clinical Investigation

Article Title: Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

doi: 10.1172/JCI181630

Figure Lengend Snippet: ( A ) Differentially expressed proteins in BAG3 –/– CFs. ( B and C ) STRING analysis of protein-protein interactions reveals 2 clusters of cell cycle– and ECM-associated protein networks. ( D ) Gene set enrichment analysis of TGF-β signaling pathway. ( E ) Fold change values for the TGF-β ligands and receptors identified by mass spectrometry. * P < 0.05 by 2-tailed Student’s t test with permutation-based FDR correction less than 0.05. n = 3 independent differentiations in mass spectrometry data.

Article Snippet: For endogenous BAG3 immunoprecipitation, 5 μg of BAG3 antibody (Proteintech 10599-1AP) was incubated with Dynabeads Protein G (25 μL) (Thermo Fisher Scientific) in TBS with 0.1% Tween-20 (TBST) for 30 minutes at room temperature.

Techniques: Protein-Protein interactions, Mass Spectrometry

( A – D ) Representative Western blot and quantification of the phosphorylation response of the canonical and non-canonical TGF-β signaling pathway following stimulation. ( E ) TGF-β pathway activity was measured by a SMAD-binding element luciferase reporter. ( F ) Silencing of TGFBR2 abrogates the hypersensitivity of BAG3 –/– reporter response. ( G ) RT-qPCR of ECM-related genes after 48 hours of TGF-β ligand stimulation following culture on tissue culture plastic (TCP). ( H ) RT-qPCR of ECM-related genes after 48 hours of TGF-β ligand stimulation following culture on 8 kPa substrate. ( I ) Western blot of FN-EDA and α-SMA response after 48 hours of TGF-β stimulation following culture on TCP. ( J ) Western blot of FN-EDA, α-SMA, and SMAD response after 48 hours of TGF-β stimulation following culture on 8 kPa substrate. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 2-way ANOVA with post hoc Šidák’s test.

Journal: The Journal of Clinical Investigation

Article Title: Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

doi: 10.1172/JCI181630

Figure Lengend Snippet: ( A – D ) Representative Western blot and quantification of the phosphorylation response of the canonical and non-canonical TGF-β signaling pathway following stimulation. ( E ) TGF-β pathway activity was measured by a SMAD-binding element luciferase reporter. ( F ) Silencing of TGFBR2 abrogates the hypersensitivity of BAG3 –/– reporter response. ( G ) RT-qPCR of ECM-related genes after 48 hours of TGF-β ligand stimulation following culture on tissue culture plastic (TCP). ( H ) RT-qPCR of ECM-related genes after 48 hours of TGF-β ligand stimulation following culture on 8 kPa substrate. ( I ) Western blot of FN-EDA and α-SMA response after 48 hours of TGF-β stimulation following culture on TCP. ( J ) Western blot of FN-EDA, α-SMA, and SMAD response after 48 hours of TGF-β stimulation following culture on 8 kPa substrate. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 2-way ANOVA with post hoc Šidák’s test.

Techniques: Western Blot, Phospho-proteomics, Activity Assay, Binding Assay, Luciferase, Quantitative RT-PCR

( A ) Identification of BAG3 binding partners in CFs using affinity purification of FLAG and mass spectrometry (AP-MS). ( B ) Immunoprecipitation of endogenous BAG3 confirms TGFBR2 interaction. ( C ) Lysosomal flux of TGFBR2 measured by Western blot. ( D ) Proteasomal flux of TGFBR2 measured by Western blot. ( E ) Quantification of C . ( F ) Quantification of D . ( G and H ) Cycloheximide (CHX) chase of V5-tagged TGFBR2 ( G ) and quantification ( H ). ( I and J ) Ubiquitination assay of V5-TGFBR2 ( I ) and quantification ( J ). ( K and L ) Rescue of TGFBR2 ubiquitination in BAG3 –/– background ( K ) and quantification ( L ). ( M ) BAG3 E455K reduces TGFBR2 and BAG3 binding. * P < 0.05, ** P < 0.01 by unpaired 2-tailed Student’s t test ( E , F , and J ), 1-way ANOVA with post hoc Tukey’s test ( L and M ), or 2-way ANOVA with post hoc Šidák’s test ( H ). n = 3 independent transfections.

Journal: The Journal of Clinical Investigation

Article Title: Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

doi: 10.1172/JCI181630

Figure Lengend Snippet: ( A ) Identification of BAG3 binding partners in CFs using affinity purification of FLAG and mass spectrometry (AP-MS). ( B ) Immunoprecipitation of endogenous BAG3 confirms TGFBR2 interaction. ( C ) Lysosomal flux of TGFBR2 measured by Western blot. ( D ) Proteasomal flux of TGFBR2 measured by Western blot. ( E ) Quantification of C . ( F ) Quantification of D . ( G and H ) Cycloheximide (CHX) chase of V5-tagged TGFBR2 ( G ) and quantification ( H ). ( I and J ) Ubiquitination assay of V5-TGFBR2 ( I ) and quantification ( J ). ( K and L ) Rescue of TGFBR2 ubiquitination in BAG3 –/– background ( K ) and quantification ( L ). ( M ) BAG3 E455K reduces TGFBR2 and BAG3 binding. * P < 0.05, ** P < 0.01 by unpaired 2-tailed Student’s t test ( E , F , and J ), 1-way ANOVA with post hoc Tukey’s test ( L and M ), or 2-way ANOVA with post hoc Šidák’s test ( H ). n = 3 independent transfections.

Techniques: Binding Assay, Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Transfection

$( A ) Uniform manifold approximation and projection (UMAP) visualization of multiplexed snRNA-Seq data of cultured CFs, color-coded based on their respective genotypes. ( B ) Averaged and normalized expression of fibrosis- and TGF-β–related genes. Median values are denoted by black horizontal bars. The interquartile range is illustrated by the upper and lower boundaries of the box. The highest and lowest values are indicated by the top and bottom ends of the vertical lines. Adjusted P values from edgeR analysis are provided at the top. ( C ) UMAP of non-failing control and DCM-affected left ventricles, distinguished by their assigned cell types. ( D ) UMAP of fibroblasts, with color indicating their respective cell states. ( E and F ) Dot plot illustrating the differentially expressed genes in BAG3 +/+ , BAG3 +/– , and BAG3 –/– hiPSC-CFs ( E ) and human tissue fibroblasts ( F ). Genes highlighted in red indicate significantly differential expression in the proteomics of BAG3 –/– hiPSC-CFs. The size of each dot represents the fraction of nuclei expressing each gene, and colors indicate log 2 fold change. Adjusted P values from edgeR analysis are displayed on top of each dot. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( G ) Representative images from staining of explanted BAG3 DCM ( n = 4) hearts and non-failing control (NFC) hearts ( n = 4) with Masson’s trichrome and Picrosirius red (PSR) staining. Scale bars: 50 μm. ( H ) Quantification of fibrosis in trichrome images. Each dot represents the average fibrosis present in patient samples from several histological slices. n = 4 for both NFC and BAG3-mutant DCM patients. ( I ) Quantification of fibrosis in PSR images. n = 4 for both NFC and BAG3-mutant DCM patients. ** P < 0.01 by 2-tailed Student’s t test ( H and I ).$

Journal: The Journal of Clinical Investigation

Article Title: Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

doi: 10.1172/JCI181630

Figure Lengend Snippet: ( A ) Uniform manifold approximation and projection (UMAP) visualization of multiplexed snRNA-Seq data of cultured CFs, color-coded based on their respective genotypes. ( B ) Averaged and normalized expression of fibrosis- and TGF-β–related genes. Median values are denoted by black horizontal bars. The interquartile range is illustrated by the upper and lower boundaries of the box. The highest and lowest values are indicated by the top and bottom ends of the vertical lines. Adjusted P values from edgeR analysis are provided at the top. ( C ) UMAP of non-failing control and DCM-affected left ventricles, distinguished by their assigned cell types. ( D ) UMAP of fibroblasts, with color indicating their respective cell states. ( E and F ) Dot plot illustrating the differentially expressed genes in BAG3 +/+ , BAG3 +/– , and BAG3 –/– hiPSC-CFs ( E ) and human tissue fibroblasts ( F ). Genes highlighted in red indicate significantly differential expression in the proteomics of BAG3 –/– hiPSC-CFs. The size of each dot represents the fraction of nuclei expressing each gene, and colors indicate log 2 fold change. Adjusted P values from edgeR analysis are displayed on top of each dot. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( G ) Representative images from staining of explanted BAG3 DCM ( n = 4) hearts and non-failing control (NFC) hearts ( n = 4) with Masson’s trichrome and Picrosirius red (PSR) staining. Scale bars: 50 μm. ( H ) Quantification of fibrosis in trichrome images. Each dot represents the average fibrosis present in patient samples from several histological slices. n = 4 for both NFC and BAG3-mutant DCM patients. ( I ) Quantification of fibrosis in PSR images. n = 4 for both NFC and BAG3-mutant DCM patients. ** P < 0.01 by 2-tailed Student’s t test ( H and I ).

Techniques: Cell Culture, Expressing, Control, Quantitative Proteomics, Staining, Mutagenesis

Figure 4. ZIPK interacts with STAT5A (A) Simulated diagram using AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region. (B) Analysis of AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region, with an interface-predicted template model (ipTM)=0.19 and a predicted template model (pTM)=0.28. (C) Simulated diagram using AlphaFold3 illustrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region. (D) Analysis of AlphaFold3 demonstrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region, with ipTM=0.27 and EpTM=0.31. (E) Simulated diagram using AlphaFold3 showing the interaction between ZIPK and STAT5A. (F) Analysis of AlphaFold3 revealed an interaction between ZIPK and STAT5A, with ipTM=0.16 and pTM=0.49. (G) The interaction between ZIPK and STAT5A in HUVECs was determined by immunoprecipitation. (H) Confocal microscopy confirmed the colocalization of ZIPK and STAT5A in HUVECs.

Journal: Acta biochimica et biophysica Sinica

Article Title: ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury.

doi: 10.3724/abbs.2024120

Figure Lengend Snippet: Figure 4. ZIPK interacts with STAT5A (A) Simulated diagram using AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region. (B) Analysis of AlphaFold3 showing the binding of ZIPK to the single-stranded DNA of the STAT5A promoter region, with an interface-predicted template model (ipTM)=0.19 and a predicted template model (pTM)=0.28. (C) Simulated diagram using AlphaFold3 illustrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region. (D) Analysis of AlphaFold3 demonstrating the binding of ZIPK to the double-stranded DNA of the STAT5A promoter region, with ipTM=0.27 and EpTM=0.31. (E) Simulated diagram using AlphaFold3 showing the interaction between ZIPK and STAT5A. (F) Analysis of AlphaFold3 revealed an interaction between ZIPK and STAT5A, with ipTM=0.16 and pTM=0.49. (G) The interaction between ZIPK and STAT5A in HUVECs was determined by immunoprecipitation. (H) Confocal microscopy confirmed the colocalization of ZIPK and STAT5A in HUVECs.

Article Snippet: Endogenous immunoprecipitation was performed by incubating cell lysates with antiZIPK antibody (1:500; 30656-1-AP; Proteintech) or anti-STAT5A antibody (1:500; 366459-1-Ig; Proteintech), using anti-IgG antibody (1:500; 30000-0-AP; Proteintech) as the negative control, supplemented with 50 μL of protein A/G Dynabeads (Thermo Fisher Scientific) at 4°C overnight.

Techniques: Binding Assay, Immunoprecipitation, Confocal Microscopy

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