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Millipore mouse anti flag m2
Mouse Anti Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore mouse anti flag m2
Mouse Anti Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore anti flag m2 affinity gel
Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore mouse monoclonal anti flag m2 antibody
The lethality of asf1Δ is suppressed by the H3-K122E mutation that disrupts the histone-Djc9 interaction. (A and B) Tetrad dissection showing that the expression of H3-K122E ( A ) but not wild-type H3 ( B ) from an integrating plasmid suppresses the lethality of asf1Δ . H3 and H4 were expressed from the same plasmid under the control of their native promoters. (C and D) Reciprocal IP showing that H3-K122E is defective in Djc9 binding. C-terminally <t>FLAG-tagged</t> wild-type H3, H3-K122E, or H3-K122R were expressed together with H4 under their native promoters in cells expressing endogenously TAP (Tandem Affinity Purification) -tagged Djc9 and endogenously 13 × Myc-tagged Asf1. The IP was performed using <t>anti-FLAG</t> <t>M2</t> beads ( C ) or IgG beads ( D ). Immunoprecipitated proteins were analyzed by immunoblotting. ( E ) In vitro Ni-NTA pull-down assay showing that H3-K122E is defective in Djc9 binding. GST-Djc9, H3-FFH, and H4 were co-expressed in E. coli . Both the pull-down and input were analyzed by SDS-PAGE and CBB staining.
Mouse Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti flag m2 antibody/product/Millipore
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Millipore anti flag m2 agarose
The lethality of asf1Δ is suppressed by the H3-K122E mutation that disrupts the histone-Djc9 interaction. (A and B) Tetrad dissection showing that the expression of H3-K122E ( A ) but not wild-type H3 ( B ) from an integrating plasmid suppresses the lethality of asf1Δ . H3 and H4 were expressed from the same plasmid under the control of their native promoters. (C and D) Reciprocal IP showing that H3-K122E is defective in Djc9 binding. C-terminally <t>FLAG-tagged</t> wild-type H3, H3-K122E, or H3-K122R were expressed together with H4 under their native promoters in cells expressing endogenously TAP (Tandem Affinity Purification) -tagged Djc9 and endogenously 13 × Myc-tagged Asf1. The IP was performed using <t>anti-FLAG</t> <t>M2</t> beads ( C ) or IgG beads ( D ). Immunoprecipitated proteins were analyzed by immunoblotting. ( E ) In vitro Ni-NTA pull-down assay showing that H3-K122E is defective in Djc9 binding. GST-Djc9, H3-FFH, and H4 were co-expressed in E. coli . Both the pull-down and input were analyzed by SDS-PAGE and CBB staining.
Anti Flag M2 Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Merck & Co mouse monoclonal anti flag m2 antibody
Hfq is required for survival during N starvation in E. coli . ( A ) Schematic showing the established mechanisms of interaction between class I and class II sRNA with Hfq and their target mRNA. Hfq monomers are represented by the six hexagons, the upper section represents the proximal RNA-binding face, the middle section represents the rim region, and the lower section represents the distal RNA-binding face. Regions of either sRNA or mRNA that associate with the different RNA-binding surfaces of Hfq are coloured according to that of the binding surfaces. ( B ) Viability of WT and Δ hfq E. coli expressing plasmid-borne Hfq (pBAD24- hfq -3xFLAG) measured by counting CFU 24 h after becoming N-starved (N-24). ( C ) Representative immunoblot of whole-cell extracts of Δ hfq E. coli containing either a pBAD18 empty vector control or pBAD24- hfq -3xFLAG, sampled at N-24. The immunoblots were probed with <t>anti-FLAG</t> antibody and anti-DnaK antibody (loading control). ( D ) Viability of WT and Δ hfq E. coli , and E. coli expressing Hfq with a C-terminal truncation from residues 73–102 ( hfq 1-72 ) measured by counting CFU at N-24. ( E ) Growth of Δ hfq E. coli expressing plasmid-borne WT or alanine mutants of Hfq (pBAD24- hfq -3xFLAG) under N limiting conditions. In panels (B), (D), and (E), error bars represent standard deviation ( n = 3). In panels (B) and (D), statistical analysis was performed by Brown–Forsyth and Welch’s ANOVA (**** P < 0.0001; ** P < 0.01).
Mouse Monoclonal Anti Flag M2 Antibody, supplied by Merck & Co, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore sigma aldrich flag m2
G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with <t>FLAG-tagged</t> G6PD. FLAG-G6PD protein was isolated using FLAG <t>M2</t> beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.
Sigma Aldrich Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore flag m2 monoclonal antibody affinity gel
G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with <t>FLAG-tagged</t> G6PD. FLAG-G6PD protein was isolated using FLAG <t>M2</t> beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.
Flag M2 Monoclonal Antibody Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Merck KGaA anti flag m2 affinity gel
G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with <t>FLAG-tagged</t> G6PD. FLAG-G6PD protein was isolated using FLAG <t>M2</t> beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.
Anti Flag M2 Affinity Gel, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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The lethality of asf1Δ is suppressed by the H3-K122E mutation that disrupts the histone-Djc9 interaction. (A and B) Tetrad dissection showing that the expression of H3-K122E ( A ) but not wild-type H3 ( B ) from an integrating plasmid suppresses the lethality of asf1Δ . H3 and H4 were expressed from the same plasmid under the control of their native promoters. (C and D) Reciprocal IP showing that H3-K122E is defective in Djc9 binding. C-terminally FLAG-tagged wild-type H3, H3-K122E, or H3-K122R were expressed together with H4 under their native promoters in cells expressing endogenously TAP (Tandem Affinity Purification) -tagged Djc9 and endogenously 13 × Myc-tagged Asf1. The IP was performed using anti-FLAG M2 beads ( C ) or IgG beads ( D ). Immunoprecipitated proteins were analyzed by immunoblotting. ( E ) In vitro Ni-NTA pull-down assay showing that H3-K122E is defective in Djc9 binding. GST-Djc9, H3-FFH, and H4 were co-expressed in E. coli . Both the pull-down and input were analyzed by SDS-PAGE and CBB staining.

Journal: Nucleic Acids Research

Article Title: The ortholog of human DNAJC9 promotes histone H3–H4 degradation and is counteracted by Asf1 in fission yeast

doi: 10.1093/nar/gkaf036

Figure Lengend Snippet: The lethality of asf1Δ is suppressed by the H3-K122E mutation that disrupts the histone-Djc9 interaction. (A and B) Tetrad dissection showing that the expression of H3-K122E ( A ) but not wild-type H3 ( B ) from an integrating plasmid suppresses the lethality of asf1Δ . H3 and H4 were expressed from the same plasmid under the control of their native promoters. (C and D) Reciprocal IP showing that H3-K122E is defective in Djc9 binding. C-terminally FLAG-tagged wild-type H3, H3-K122E, or H3-K122R were expressed together with H4 under their native promoters in cells expressing endogenously TAP (Tandem Affinity Purification) -tagged Djc9 and endogenously 13 × Myc-tagged Asf1. The IP was performed using anti-FLAG M2 beads ( C ) or IgG beads ( D ). Immunoprecipitated proteins were analyzed by immunoblotting. ( E ) In vitro Ni-NTA pull-down assay showing that H3-K122E is defective in Djc9 binding. GST-Djc9, H3-FFH, and H4 were co-expressed in E. coli . Both the pull-down and input were analyzed by SDS-PAGE and CBB staining.

Article Snippet: The antibodies used for immunoblotting were: a rat polyclonal antibody against Djc9 and a rat polyclonal antibody against Asf1 generated in this study; rabbit polyclonal anti-H2A antibody (GeneTeX, GTX129418), rabbit polyclonal anti-H2B antibody (GeneTeX, GTX129565), rabbit polyclonal anti-H3 antibody (Abcam, ab1791); rabbit polyclonal anti-H4 antibody (Abcam, ab10158); rabbit polyclonal anti-H3K56ac antibody (Sigma, 07–677-IS); mouse monoclonal anti-mini-AID tag antibody (MBL, M214-3); mouse monoclonal anti-FLAG M2 antibody (Sigma, F1804); mouse monoclonal anti-GFP antibody (Roche, 11 814 460 001); mouse monoclonal anti-c-Myc antibody (Roche, 11 667 203 001); monoclonal anti-GST-HRP (Sigma, A7304); peroxidase anti-peroxidase (PAP) antibody for detecting TAP-tagged proteins (Sigma, P1291); rabbit polyclonal anti-Lap2 antibody (Du lab stock).

Techniques: Mutagenesis, Dissection, Expressing, Plasmid Preparation, Control, Binding Assay, Affinity Purification, Immunoprecipitation, Western Blot, In Vitro, Pull Down Assay, SDS Page, Staining

Djc9 confers HU resistance by promoting H3–H4 degradation. ( A ) djc9Δ cells exhibited sensitivity to the DNA replication inhibitor HU. Five-fold dilutions of cells were spotted on YES plates and YES plates containing HU, CPT, or MMS. Plates were scanned after 2 days of incubation at 30°C. ( B ) The levels of histones H3 and H4 were reduced upon HU treatment in a Djc9-dependent manner. The levels of endogenous H3 and H4 were examined by immunoblotting analysis of whole cell extracts of wild-type and djc9Δ cells treated or not treated with 15 mM of HU. Two clones were analyzed for each genotype. ( C ) Immunoblotting with H3K4me3-specific antibodies showing that pre-existing histones with H3K4me3 modification decreased after HU treatment in both wild-type and djc9Δ cells. ( D ) Immunoblotting showing that newly synthesized FLAG-tagged H3 from the sucrose-inducible Pinv1 promoter accumulated in HU-arrested djc9Δ cells but not in HU-arrested wild-type cells. This difference was abolished by the proteasome inhibitor BTZ. Cells grown in 8% glucose medium were pre-treated with HU for 4 h and then shifted to HU-containing 4% sucrose medium with or without BTZ. Cells were collected 1 h after the shift and whole cell extracts were analyzed using immunoblotting with antibodies against FLAG, Djc9, and H3. ( E ) Reducing the dosage of H3 and H4 genes suppressed the HU sensitivity of djc9Δ cells. The growth phenotype of strains with indicated genotypes was analyzed using a spot assay.

Journal: Nucleic Acids Research

Article Title: The ortholog of human DNAJC9 promotes histone H3–H4 degradation and is counteracted by Asf1 in fission yeast

doi: 10.1093/nar/gkaf036

Figure Lengend Snippet: Djc9 confers HU resistance by promoting H3–H4 degradation. ( A ) djc9Δ cells exhibited sensitivity to the DNA replication inhibitor HU. Five-fold dilutions of cells were spotted on YES plates and YES plates containing HU, CPT, or MMS. Plates were scanned after 2 days of incubation at 30°C. ( B ) The levels of histones H3 and H4 were reduced upon HU treatment in a Djc9-dependent manner. The levels of endogenous H3 and H4 were examined by immunoblotting analysis of whole cell extracts of wild-type and djc9Δ cells treated or not treated with 15 mM of HU. Two clones were analyzed for each genotype. ( C ) Immunoblotting with H3K4me3-specific antibodies showing that pre-existing histones with H3K4me3 modification decreased after HU treatment in both wild-type and djc9Δ cells. ( D ) Immunoblotting showing that newly synthesized FLAG-tagged H3 from the sucrose-inducible Pinv1 promoter accumulated in HU-arrested djc9Δ cells but not in HU-arrested wild-type cells. This difference was abolished by the proteasome inhibitor BTZ. Cells grown in 8% glucose medium were pre-treated with HU for 4 h and then shifted to HU-containing 4% sucrose medium with or without BTZ. Cells were collected 1 h after the shift and whole cell extracts were analyzed using immunoblotting with antibodies against FLAG, Djc9, and H3. ( E ) Reducing the dosage of H3 and H4 genes suppressed the HU sensitivity of djc9Δ cells. The growth phenotype of strains with indicated genotypes was analyzed using a spot assay.

Article Snippet: The antibodies used for immunoblotting were: a rat polyclonal antibody against Djc9 and a rat polyclonal antibody against Asf1 generated in this study; rabbit polyclonal anti-H2A antibody (GeneTeX, GTX129418), rabbit polyclonal anti-H2B antibody (GeneTeX, GTX129565), rabbit polyclonal anti-H3 antibody (Abcam, ab1791); rabbit polyclonal anti-H4 antibody (Abcam, ab10158); rabbit polyclonal anti-H3K56ac antibody (Sigma, 07–677-IS); mouse monoclonal anti-mini-AID tag antibody (MBL, M214-3); mouse monoclonal anti-FLAG M2 antibody (Sigma, F1804); mouse monoclonal anti-GFP antibody (Roche, 11 814 460 001); mouse monoclonal anti-c-Myc antibody (Roche, 11 667 203 001); monoclonal anti-GST-HRP (Sigma, A7304); peroxidase anti-peroxidase (PAP) antibody for detecting TAP-tagged proteins (Sigma, P1291); rabbit polyclonal anti-Lap2 antibody (Du lab stock).

Techniques: Incubation, Western Blot, Clone Assay, Modification, Synthesized, Spot Test

Hfq is required for survival during N starvation in E. coli . ( A ) Schematic showing the established mechanisms of interaction between class I and class II sRNA with Hfq and their target mRNA. Hfq monomers are represented by the six hexagons, the upper section represents the proximal RNA-binding face, the middle section represents the rim region, and the lower section represents the distal RNA-binding face. Regions of either sRNA or mRNA that associate with the different RNA-binding surfaces of Hfq are coloured according to that of the binding surfaces. ( B ) Viability of WT and Δ hfq E. coli expressing plasmid-borne Hfq (pBAD24- hfq -3xFLAG) measured by counting CFU 24 h after becoming N-starved (N-24). ( C ) Representative immunoblot of whole-cell extracts of Δ hfq E. coli containing either a pBAD18 empty vector control or pBAD24- hfq -3xFLAG, sampled at N-24. The immunoblots were probed with anti-FLAG antibody and anti-DnaK antibody (loading control). ( D ) Viability of WT and Δ hfq E. coli , and E. coli expressing Hfq with a C-terminal truncation from residues 73–102 ( hfq 1-72 ) measured by counting CFU at N-24. ( E ) Growth of Δ hfq E. coli expressing plasmid-borne WT or alanine mutants of Hfq (pBAD24- hfq -3xFLAG) under N limiting conditions. In panels (B), (D), and (E), error bars represent standard deviation ( n = 3). In panels (B) and (D), statistical analysis was performed by Brown–Forsyth and Welch’s ANOVA (**** P < 0.0001; ** P < 0.01).

Journal: Nucleic Acids Research

Article Title: Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

doi: 10.1093/nar/gkaf019

Figure Lengend Snippet: Hfq is required for survival during N starvation in E. coli . ( A ) Schematic showing the established mechanisms of interaction between class I and class II sRNA with Hfq and their target mRNA. Hfq monomers are represented by the six hexagons, the upper section represents the proximal RNA-binding face, the middle section represents the rim region, and the lower section represents the distal RNA-binding face. Regions of either sRNA or mRNA that associate with the different RNA-binding surfaces of Hfq are coloured according to that of the binding surfaces. ( B ) Viability of WT and Δ hfq E. coli expressing plasmid-borne Hfq (pBAD24- hfq -3xFLAG) measured by counting CFU 24 h after becoming N-starved (N-24). ( C ) Representative immunoblot of whole-cell extracts of Δ hfq E. coli containing either a pBAD18 empty vector control or pBAD24- hfq -3xFLAG, sampled at N-24. The immunoblots were probed with anti-FLAG antibody and anti-DnaK antibody (loading control). ( D ) Viability of WT and Δ hfq E. coli , and E. coli expressing Hfq with a C-terminal truncation from residues 73–102 ( hfq 1-72 ) measured by counting CFU at N-24. ( E ) Growth of Δ hfq E. coli expressing plasmid-borne WT or alanine mutants of Hfq (pBAD24- hfq -3xFLAG) under N limiting conditions. In panels (B), (D), and (E), error bars represent standard deviation ( n = 3). In panels (B) and (D), statistical analysis was performed by Brown–Forsyth and Welch’s ANOVA (**** P < 0.0001; ** P < 0.01).

Article Snippet: For immunoblotting of Hfq protein, mouse monoclonal Anti-FLAG ® M2 antibody (Merck, F1804) was used at 1:1000 dilution.

Techniques: RNA Binding Assay, Binding Assay, Expressing, Plasmid Preparation, Western Blot, Control, Standard Deviation

Characterization of a systematic alanine mutant library of Hfq. ( A ) Viability of WT and Δ hfq E. coli expressing plasmid-borne alanine mutants of Hfq (pBAD24- hfq -3xFLAG) measured by counting CFU following N-24. Values are represented as a percentage of viable cell counts relative to the WT complemented strain. The dashed red line indicates ≥25% drop in viable cell count. Error bars represent standard deviation ( n = 3). Statistical analysis performed by Brown–Forsyth and Welch’s ANOVA. (*** P < 0.001; ** P < 0.01; * P < 0.05). ( B) Structures of hexameric Hfq (PDB: 1HK9) with each monomer of Hfq coloured separately. The aa residues at which an alanine substitution results in ≥25% decrease in viability with respect to WT (black bars in panel A) are labelled. The aa residues at or close to the proximal face and distal face that are surface exposed (marked with *) or not fully buried within the Hfq hexamer are shown in red and orange, respectively; the surface-exposed rim residues are shown in blue; and residues fully buried and internal to the Hfq hexamer are shown in purple. Residues at which alanine substitution resulted in >50% reduction in protein expression are underlined (see panel C). Inset shows a magnified view of residues found at internally or at the monomer–monomer interfaces, with the residue sidechains shown. Note that aa K3 is not present in the Hfq structure used here. ( C ) Representative immunoblots of whole-cell extracts of Δ hfq E. coli containing either a pBAD18 empty vector control, or WT or alanine mutants of pBAD24- hfq -3xFLAG, sampled at N-24. The immunoblots were probed with anti-FLAG antibody and anti-DnaK antibody (loading control). The ratio of the intensity of bands corresponding to alanine mutants of Hfq relative to that of WT Hfq from the same immunoblot are indicated below the immunoblot.

Journal: Nucleic Acids Research

Article Title: Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

doi: 10.1093/nar/gkaf019

Figure Lengend Snippet: Characterization of a systematic alanine mutant library of Hfq. ( A ) Viability of WT and Δ hfq E. coli expressing plasmid-borne alanine mutants of Hfq (pBAD24- hfq -3xFLAG) measured by counting CFU following N-24. Values are represented as a percentage of viable cell counts relative to the WT complemented strain. The dashed red line indicates ≥25% drop in viable cell count. Error bars represent standard deviation ( n = 3). Statistical analysis performed by Brown–Forsyth and Welch’s ANOVA. (*** P < 0.001; ** P < 0.01; * P < 0.05). ( B) Structures of hexameric Hfq (PDB: 1HK9) with each monomer of Hfq coloured separately. The aa residues at which an alanine substitution results in ≥25% decrease in viability with respect to WT (black bars in panel A) are labelled. The aa residues at or close to the proximal face and distal face that are surface exposed (marked with *) or not fully buried within the Hfq hexamer are shown in red and orange, respectively; the surface-exposed rim residues are shown in blue; and residues fully buried and internal to the Hfq hexamer are shown in purple. Residues at which alanine substitution resulted in >50% reduction in protein expression are underlined (see panel C). Inset shows a magnified view of residues found at internally or at the monomer–monomer interfaces, with the residue sidechains shown. Note that aa K3 is not present in the Hfq structure used here. ( C ) Representative immunoblots of whole-cell extracts of Δ hfq E. coli containing either a pBAD18 empty vector control, or WT or alanine mutants of pBAD24- hfq -3xFLAG, sampled at N-24. The immunoblots were probed with anti-FLAG antibody and anti-DnaK antibody (loading control). The ratio of the intensity of bands corresponding to alanine mutants of Hfq relative to that of WT Hfq from the same immunoblot are indicated below the immunoblot.

Article Snippet: For immunoblotting of Hfq protein, mouse monoclonal Anti-FLAG ® M2 antibody (Merck, F1804) was used at 1:1000 dilution.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Cell Counting, Standard Deviation, Residue, Western Blot, Control

G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with FLAG-tagged G6PD. FLAG-G6PD protein was isolated using FLAG M2 beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.

Journal: Life Metabolism

Article Title: Glucose-6-phosphate dehydrogenase regulates mitophagy by maintaining PINK1 stability

doi: 10.1093/lifemeta/loae040

Figure Lengend Snippet: G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with FLAG-tagged G6PD. FLAG-G6PD protein was isolated using FLAG M2 beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.

Article Snippet: The following primary antibodies were used: Cell Signaling Technology: AMPKα (#5832), GFP (#2956), SAPK/JNK (#9252), K48-linkage Specific Polyubiquitin (#8081), Mitofusin-1 (#14739), Mitofusin-2 (#11925), phospho-AMPKα (Thr172) (#2535), phospho-SAPK/JNK (Thr183/Tyr185) (#4668), phospho-Ubiquitin (Ser65) (#62802), Parkin (#4211), PINK1 (#6946), and TOM20 (#42406); Santa Cruz Biotechnology: G6PD (sc-373886) and HA tag (sc-7392); Sigma-Aldrich: FLAG® M2 (F1804), α-Tubulin (T6199), and β-Actin (A5441); Abcam: GAPDH (ab8245), mCherry (ab167453), and MTCO2 (COXII) (ab110258); BD Transduction Laboratories: TIM23 (611222); Proteintech: VDAC1/2 (10866-1-AP).

Techniques: Western Blot, Fractionation, Gradient Centrifugation, Marker, Immunoprecipitation, Transfection, Lysis, In Vitro, Pull Down Assay, Isolation, Incubation, Recombinant, SDS Page

G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with FLAG-tagged G6PD. FLAG-G6PD protein was isolated using FLAG M2 beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.

Journal: Life Metabolism

Article Title: Glucose-6-phosphate dehydrogenase regulates mitophagy by maintaining PINK1 stability

doi: 10.1093/lifemeta/loae040

Figure Lengend Snippet: G6PD interacts with the mitophagy machinery. (a) Mitochondrial G6PD levels detected by immunoblotting analysis. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 1 h or 2 h. Cells were subjected to fractionation by sucrose gradient centrifugation and immunoblotted with the indicated antibodies. Sample loading was standardized to the whole cell lysate. GAPDH, cytosolic marker; TIM23, mitochondrial marker. WCL, whole cell lysate; Cyto, cytosol; Mito, mitochondria. (b) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and TOM20 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (c) Visualization of G6PD mitochondrial localization by proteinase K protection assay. HeLa 3+ cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and subjected to fractionation by sucrose gradient centrifugation. The mitochondrial fraction was divided and treated with the indicated concentrations of digitonin and proteinase K. Samples were then subjected to immunoblotting analysis. (d) PLA performed on YFP-Parkin HeLa cells. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min. Assay was performed using G6PD and PINK1 primary antibodies. Red, PLA signal; gray (pseudocoloured), YFP-Parkin; blue, DAPI. Scale bar: 20 μm. (e) Immunoprecipitation of overexpressed myc-PINK1 with endogenous G6PD. HeLa 3+ cells were transfected with myc-PINK1 for 24 h. Cells were treated with O/A (5 μmol/L and 1 μmol/L, respectively) for 90 min and lysed in IP lysis buffer. Lysates were subjected to IP with agarose-conjugated G6PD antibody and blotted with the indicated antibodies. (f) In vitro pull-down assay between G6PD and PINK1. HeLa mCherry-Parkin cells were transfected with FLAG-tagged G6PD. FLAG-G6PD protein was isolated using FLAG M2 beads and incubated with recombinant human PINK1 protein. Samples were subjected to SDS-PAGE and western blotting analysis to visualize pull-down of PINK1.

Article Snippet: FLAG-G6PD was purified by pull-down with FLAG M2 monoclonal antibody affinity gel (Sigma-Aldrich, A2220).

Techniques: Western Blot, Fractionation, Gradient Centrifugation, Marker, Immunoprecipitation, Transfection, Lysis, In Vitro, Pull Down Assay, Isolation, Incubation, Recombinant, SDS Page