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anti rodent bnip3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti rodent bnip3
    (A, B) Western blots of <t>BNIP3</t> (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.
    Anti Rodent Bnip3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rodent bnip3/product/Cell Signaling Technology Inc
    Average 96 stars, based on 337 article reviews
    anti rodent bnip3 - by Bioz Stars, 2026-06
    96/100 stars

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    1) Product Images from "Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX"

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202402765

    (A, B) Western blots of BNIP3 (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.
    Figure Legend Snippet: (A, B) Western blots of BNIP3 (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.

    Techniques Used: Western Blot, Control, Quantitative RT-PCR, Fluorescence

    (A) Analysis of select HIF-responsive targets in Pptc7 KO heart and liver proteomics datasets collected in . (B, C) Western blots of exogenous BNIP3 (B) or NIX (C) expressed in wild-type PPTC7 KO 293T cells at various plasmid concentrations. “D” indicates dimer species, “M” indicates monomer species. Actin shown as a loading control. (D, E) FACS analysis of mt-Keima positive wild-type (D) and Pptc7 KO (E) MEFs showing high mitophagy rates in response to variable DFO concentrations. (D, E, F) Statistical analysis of data by ordinary one-way ANOVA shown in (D, E), gray bars = wild-type samples, pink bars = Pptc7 KO samples. **** P < 0.0001, *** P < 0.001, ** P < 0.01, ns = not significant. Error bars represent SD, data points represent individual biological replicates (n = 3).
    Figure Legend Snippet: (A) Analysis of select HIF-responsive targets in Pptc7 KO heart and liver proteomics datasets collected in . (B, C) Western blots of exogenous BNIP3 (B) or NIX (C) expressed in wild-type PPTC7 KO 293T cells at various plasmid concentrations. “D” indicates dimer species, “M” indicates monomer species. Actin shown as a loading control. (D, E) FACS analysis of mt-Keima positive wild-type (D) and Pptc7 KO (E) MEFs showing high mitophagy rates in response to variable DFO concentrations. (D, E, F) Statistical analysis of data by ordinary one-way ANOVA shown in (D, E), gray bars = wild-type samples, pink bars = Pptc7 KO samples. **** P < 0.0001, *** P < 0.001, ** P < 0.01, ns = not significant. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Techniques Used: Western Blot, Plasmid Preparation, Control

    (A) DepMap essentiality profiles of PPTC7 gene effect (x-axis) and FBXL4 gene effect (y-axis). Linear regression analysis and associated P -value shown. (B, C, D, E) Statistical analysis of BNIP3 and NIX monomer and dimer turnover rate data in wild-type (gray bars) and PPTC7 KO (red bards) 293T cells shown in . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD, data points represent individual biological replicates (n = 3).
    Figure Legend Snippet: (A) DepMap essentiality profiles of PPTC7 gene effect (x-axis) and FBXL4 gene effect (y-axis). Linear regression analysis and associated P -value shown. (B, C, D, E) Statistical analysis of BNIP3 and NIX monomer and dimer turnover rate data in wild-type (gray bars) and PPTC7 KO (red bards) 293T cells shown in . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Techniques Used:

    (A) Schematic of DFO treatment and washout timeline and mechanism; figure made with BioRender. (B) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout, when applicable. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (C) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (C, D) Quantification of data shown in (C). BNIP3 monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (E) Western blot of endogenous NIX protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (E, F) Quantification of data shown in (E). NIX monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (G) Western blots of endogenous BNIP3 (top panel) and NIX (bottom panel) in wild-type and PPTC7 KO 293T cells upon treatment with 10 μM MG-132 for the indicated timeframes. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (G, H) Quantification of BNIP3 and NIX monomer (left graphs) and dimer (right graphs) populations in wild-type (gray) and PPTC7 KO (pink) cells shown in (G). Bands were quantified using densitometry, averaged, and plotted over time. Data were analyzed via linear regression, and the significance between slopes was calculated using Analysis of Covariance (ANCOVA). (I) Western blot of endogenous BNIP3 and NIX proteins in wild-type and PPTC7 KO cells after DFO treatment and subsequent washout in the presence or absence of 10 μM MG-132. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control.
    Figure Legend Snippet: (A) Schematic of DFO treatment and washout timeline and mechanism; figure made with BioRender. (B) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout, when applicable. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (C) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (C, D) Quantification of data shown in (C). BNIP3 monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (E) Western blot of endogenous NIX protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (E, F) Quantification of data shown in (E). NIX monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (G) Western blots of endogenous BNIP3 (top panel) and NIX (bottom panel) in wild-type and PPTC7 KO 293T cells upon treatment with 10 μM MG-132 for the indicated timeframes. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (G, H) Quantification of BNIP3 and NIX monomer (left graphs) and dimer (right graphs) populations in wild-type (gray) and PPTC7 KO (pink) cells shown in (G). Bands were quantified using densitometry, averaged, and plotted over time. Data were analyzed via linear regression, and the significance between slopes was calculated using Analysis of Covariance (ANCOVA). (I) Western blot of endogenous BNIP3 and NIX proteins in wild-type and PPTC7 KO cells after DFO treatment and subsequent washout in the presence or absence of 10 μM MG-132. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control.

    Techniques Used: Western Blot, Control

    (A) Representative single-plane confocal images of GFP only (top panels) or PPTC7-GFP (bottom panels) expressed in HeLa cells treated for 12 h with 500 μM CoCl 2 . Cells were stained for BNIP3 (second column) or TOMM20 (third column). Overlays are shown for GFP and TOMM20 (fourth column) and GFP and BNIP3 (fifth column). Scale bar = 20 μm. (A, B) Quantification of data shown in (A), mitochondrial BNIP3 staining versus non-specific staining in cells overexpressing GFP only or PPTC7-GFP versus matched untransfected controls for each experiment. Error bars represent the standard error of the mean of three independent experiments. (C) Schematic of PPTC7 features, including a mitochondrial targeting sequence (MTS) and PP2C phosphatase domain, top. Bottom, AlphaFold2 representation of PPTC7 structure; predicted disordered N-terminus and PP2C phosphatase domains indicated. (D) Western blot of 293T cells overexpressing PPTC7 or a ΔMTS-PPTC7 mutant. Red arrows indicate dual species in wild-type PPTC7 expression. * represents a non-specific band. Actin shown as a loading control. (E) PPTC7 protease protection assay. Mitochondria from HeLa FLP-IN cells expressing PPTC7 were isolated and treated with SDS, proteinase K, or both and resolved via SDS–PAGE. Western blots of PPTC7 (top), the outer mitochondrial membrane marker NIX (middle) and the matrix marker TFAM (bottom) shown. (F, G, H) Western blots of BNIP3, NIX, and PPTC7 depicting the ability of various mutants of PPTC7 to suppress BNIP3 and NIX accumulation in response to CoCl 2 treatment. “D” indicates dimer species, “M” indicates monomer species. In (F), a catalytic mutant of PPTC7, D78A, cannot effectively suppress BNIP3 and NIX accumulation relative to wild-type PPTC7. In (G), the ΔMTS-PPTC7 mutant partially or fully suppresses BNIP3 and NIX accumulation, respectively. In (H), a mutant that artificially anchors PPTC7 to the outer mitochondrial membrane, ΔMTS-PPTC7-OMP25, suppresses BNIP3 and NIX accumulation. Actin shown as a loading control.
    Figure Legend Snippet: (A) Representative single-plane confocal images of GFP only (top panels) or PPTC7-GFP (bottom panels) expressed in HeLa cells treated for 12 h with 500 μM CoCl 2 . Cells were stained for BNIP3 (second column) or TOMM20 (third column). Overlays are shown for GFP and TOMM20 (fourth column) and GFP and BNIP3 (fifth column). Scale bar = 20 μm. (A, B) Quantification of data shown in (A), mitochondrial BNIP3 staining versus non-specific staining in cells overexpressing GFP only or PPTC7-GFP versus matched untransfected controls for each experiment. Error bars represent the standard error of the mean of three independent experiments. (C) Schematic of PPTC7 features, including a mitochondrial targeting sequence (MTS) and PP2C phosphatase domain, top. Bottom, AlphaFold2 representation of PPTC7 structure; predicted disordered N-terminus and PP2C phosphatase domains indicated. (D) Western blot of 293T cells overexpressing PPTC7 or a ΔMTS-PPTC7 mutant. Red arrows indicate dual species in wild-type PPTC7 expression. * represents a non-specific band. Actin shown as a loading control. (E) PPTC7 protease protection assay. Mitochondria from HeLa FLP-IN cells expressing PPTC7 were isolated and treated with SDS, proteinase K, or both and resolved via SDS–PAGE. Western blots of PPTC7 (top), the outer mitochondrial membrane marker NIX (middle) and the matrix marker TFAM (bottom) shown. (F, G, H) Western blots of BNIP3, NIX, and PPTC7 depicting the ability of various mutants of PPTC7 to suppress BNIP3 and NIX accumulation in response to CoCl 2 treatment. “D” indicates dimer species, “M” indicates monomer species. In (F), a catalytic mutant of PPTC7, D78A, cannot effectively suppress BNIP3 and NIX accumulation relative to wild-type PPTC7. In (G), the ΔMTS-PPTC7 mutant partially or fully suppresses BNIP3 and NIX accumulation, respectively. In (H), a mutant that artificially anchors PPTC7 to the outer mitochondrial membrane, ΔMTS-PPTC7-OMP25, suppresses BNIP3 and NIX accumulation. Actin shown as a loading control.

    Techniques Used: Staining, Sequencing, Western Blot, Mutagenesis, Expressing, Control, Isolation, SDS Page, Membrane, Marker

    (A) FACS data shown for data represented in . Cells undergoing high mitophagy are above the diagonal line; percentages indicated in the figure. (B) Quantification of mt-Keima positive mitophagic flux in HeLa FLP-IN TREx cells expressing vector only (left) or PPTC7-FLAG in the presence of 10 μM doxycycline (dox, to promote PPTC7 expression), 200 μM cobalt chloride (CoCl 2 ), or both. ** P < 0.01, ns = not significant, ordinary one-way ANOVA. Error bars represent SD. Each dot represents an independent biological replicate (n = 3). (C) Western blot of PPTC7 expressed in wild-type MEFs (black arrows), Pptc7 KO MEFs, or Pptc7 KO MEFs rescued with human PPTC7 (red arrows). * represents a non-specific band. Basal BNIP3 levels across samples shown below; actin shown as a loading control. (D) Western blot of BNIP3 in crude mitochondria isolated from wild-type or PPTC7 KO 293T cells. Mitochondria were left untreated, treated with recombinant PPTC7, or treated with recombinant PPTC7 D78A. Revert stain is shown for loading; equal loading of recombinant proteins can be seen as depicted by arrows. (E) Alphafold2 model of PPTC7-NIX interaction suggesting PPTC7 D78 and NIX S146 as binding sites. (F) Western blot of NIX monomer immunoprecipitation with FLAG-tagged PPTC7 WT and PPTC7 D78A in whole cell extract. Whole-cell extracts were subjected to immunoprecipitation with anti-FLAG agarose beads and immunoblotted as indicated. Revert stain shown as a loading control.
    Figure Legend Snippet: (A) FACS data shown for data represented in . Cells undergoing high mitophagy are above the diagonal line; percentages indicated in the figure. (B) Quantification of mt-Keima positive mitophagic flux in HeLa FLP-IN TREx cells expressing vector only (left) or PPTC7-FLAG in the presence of 10 μM doxycycline (dox, to promote PPTC7 expression), 200 μM cobalt chloride (CoCl 2 ), or both. ** P < 0.01, ns = not significant, ordinary one-way ANOVA. Error bars represent SD. Each dot represents an independent biological replicate (n = 3). (C) Western blot of PPTC7 expressed in wild-type MEFs (black arrows), Pptc7 KO MEFs, or Pptc7 KO MEFs rescued with human PPTC7 (red arrows). * represents a non-specific band. Basal BNIP3 levels across samples shown below; actin shown as a loading control. (D) Western blot of BNIP3 in crude mitochondria isolated from wild-type or PPTC7 KO 293T cells. Mitochondria were left untreated, treated with recombinant PPTC7, or treated with recombinant PPTC7 D78A. Revert stain is shown for loading; equal loading of recombinant proteins can be seen as depicted by arrows. (E) Alphafold2 model of PPTC7-NIX interaction suggesting PPTC7 D78 and NIX S146 as binding sites. (F) Western blot of NIX monomer immunoprecipitation with FLAG-tagged PPTC7 WT and PPTC7 D78A in whole cell extract. Whole-cell extracts were subjected to immunoprecipitation with anti-FLAG agarose beads and immunoblotted as indicated. Revert stain shown as a loading control.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Control, Isolation, Recombinant, Staining, Binding Assay, Immunoprecipitation

    (A) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with or without 24-h deferoxamine (DFO) treatment. PPTC7-V5-miniTurbo, as well as vector only or V5-miniTurbo-only controls, were transfected into 293T cells. Streptavidin pulldowns were used to enrich for PPTC7-V5-miniTurbo interactors, which were run on SDS–PAGE gels and western blotted for BNIP3 (top blot) or NIX (second blot). Only PPTC7-V5-miniTurbo + biotin samples pulled down BNIP3 and NIX (lanes 6 and 12, streptavidin pull-down gels), indicating specific binding. Western blots shown for reaction input for pulldowns for V5 (showing miniTurbo constructs), BNIP3, NIX, and actin (serving as a load control). (B) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with after 24 h DFO treatment with or without 4 h DFO washout. Streptavidin pulldowns were used to enrich PPTC7-V5-miniTurbo interactors as described in (A). Western blots shown for reaction input for pulldowns as described in (A). (C) A representative maximum z-projection confocal image (left) and corresponding single plane insets (right) are shown of a U2OS cell overexpressing PPTC7-GFP and treated with deferiprone for 24 h. Cells were fixed and stained for BNIP3 and TOMM20 to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 449). (D) As in (C) for cells treated for 24 h with deferiprone and washed for an additional 4 h before fixation. Cells were stained to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 526).
    Figure Legend Snippet: (A) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with or without 24-h deferoxamine (DFO) treatment. PPTC7-V5-miniTurbo, as well as vector only or V5-miniTurbo-only controls, were transfected into 293T cells. Streptavidin pulldowns were used to enrich for PPTC7-V5-miniTurbo interactors, which were run on SDS–PAGE gels and western blotted for BNIP3 (top blot) or NIX (second blot). Only PPTC7-V5-miniTurbo + biotin samples pulled down BNIP3 and NIX (lanes 6 and 12, streptavidin pull-down gels), indicating specific binding. Western blots shown for reaction input for pulldowns for V5 (showing miniTurbo constructs), BNIP3, NIX, and actin (serving as a load control). (B) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with after 24 h DFO treatment with or without 4 h DFO washout. Streptavidin pulldowns were used to enrich PPTC7-V5-miniTurbo interactors as described in (A). Western blots shown for reaction input for pulldowns as described in (A). (C) A representative maximum z-projection confocal image (left) and corresponding single plane insets (right) are shown of a U2OS cell overexpressing PPTC7-GFP and treated with deferiprone for 24 h. Cells were fixed and stained for BNIP3 and TOMM20 to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 449). (D) As in (C) for cells treated for 24 h with deferiprone and washed for an additional 4 h before fixation. Cells were stained to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 526).

    Techniques Used: Labeling, Plasmid Preparation, Transfection, SDS Page, Western Blot, Binding Assay, Construct, Control, Staining



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    (A, B) Western blots of <t>BNIP3</t> (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.
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    96
    Cell Signaling Technology Inc rodent specific bnip3
    (A, B) Western blots of <t>BNIP3</t> (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.
    Rodent Specific Bnip3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rodent  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc rodent
    (A, B) Western blots of <t>BNIP3</t> (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.
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    Figure 3. <t>BNIP3</t> enhances mt-reactive oxygen species production, and attenuates mt-membrane
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    Cell Signaling Technology Inc bnip3 rodent
    Validation of the LV and RV proteomic findings. ( A ). “Core Analyses” was performed in IPA for each of the two-group comparisons, i.e., ShLuc vs. Sham and ShBNIP3 vs. ShLuc, with a cutoff p -value < 0.05. Analyzed datasets were then compared with each other in IPA’s “Comparison Analyses” function to yield the most enriched Canonical Metabolic Pathways (left) and Upstream Regulators (right) that were shared among the two-group comparisons. The respective z-score-based heat maps indicate shared Canonical Pathways and Upstream Regulators that were upregulated/activated or downregulated/inhibited in the two-group comparisons, with orange and blue color intensities representing the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. ( B ). Oxidative phosphorylation (OXPHOS) network of the electron transport chain (ETC) complexes I–V transport system presented in ShLuc vs. Sham (upper panel) and ShBNIP3 vs. ShLuc (lower panel) in LV (left) and RV (right) proteomic datasets. The network highlights mt-OXPHOS proteins in each ETC complex that were downregulated (green) or upregulated (red) or were unchanged (grey) within the two-group comparisons, and those that were not identified (white) in the LV and RV proteomic datasets. The darker the green or red color, the higher the degree of decrease or increase in relative expression, respectively. ( C , D ). Expression of the ETC complexes I–V, <t>BNIP3,</t> and molecular markers involved in mt-biogenesis and metabolism by western blot, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. Abbreviations: ShL, ShLuc; ShB, ShBNIP3.
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    Image Search Results


    (A, B) Western blots of BNIP3 (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A, B) Western blots of BNIP3 (top panels), NIX (middle panels) and actin (serving as a load control, bottom panels) in wild-type or Pptc7 KO MEFs (A) or in wild-type or PPTC7 KO 293T cells (B). “D” indicates dimer species; “M” indicates monomer species. (C) Western blot for HIF-1α in untreated (UT), vehicle only (veh, 0.2% DMSO) or 100 nM bafilomycin A (BafA) for 16 h in wild-type and PPTC7 KO 293T cells. Actin shown as a loading control. (D) qRT-PCR of BNIP3 and BNIP3L (gene name of NIX) endogenous mRNA levels in wild-type (gray) and PPTC7 KO (pink) 293T cells. Error bars represent SD; data points represent independent experiments. (E) qRT-PCR of BNIP3 RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (F) Western blotting for endogenous BNIP3 levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (G) qRT-PCR of BNIP3L RNA levels in untreated and DFO-treated (100 μM, 24 h) wild-type (gray) and PPTC7 KO (pink) 293T cells. ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent independent experiments. (H) Western blotting for endogenous NIX levels in wild-type or PPTC7 KO 293T cells treated with 100 μM DFO for indicated times. Actin shown as a loading control. (I) FACS plots of basal mitophagy in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. Cells undergoing high mitophagy are above the diagonal line; percentages indicated in figure. (J) FACS plots of mitophagy rates upon 24 h of 100 μM DFO treatment in wild-type (left), Pptc7 KO (middle), and Pptc7/Bnip3/Bnip3l TKO MEFs using the mt-Keima fluorescence assay. (I, J, K) Quantification of mt-Keima data shown in (I, J). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD; data points represent individual biological replicates.

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Western Blot, Control, Quantitative RT-PCR, Fluorescence

    (A) Analysis of select HIF-responsive targets in Pptc7 KO heart and liver proteomics datasets collected in . (B, C) Western blots of exogenous BNIP3 (B) or NIX (C) expressed in wild-type PPTC7 KO 293T cells at various plasmid concentrations. “D” indicates dimer species, “M” indicates monomer species. Actin shown as a loading control. (D, E) FACS analysis of mt-Keima positive wild-type (D) and Pptc7 KO (E) MEFs showing high mitophagy rates in response to variable DFO concentrations. (D, E, F) Statistical analysis of data by ordinary one-way ANOVA shown in (D, E), gray bars = wild-type samples, pink bars = Pptc7 KO samples. **** P < 0.0001, *** P < 0.001, ** P < 0.01, ns = not significant. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) Analysis of select HIF-responsive targets in Pptc7 KO heart and liver proteomics datasets collected in . (B, C) Western blots of exogenous BNIP3 (B) or NIX (C) expressed in wild-type PPTC7 KO 293T cells at various plasmid concentrations. “D” indicates dimer species, “M” indicates monomer species. Actin shown as a loading control. (D, E) FACS analysis of mt-Keima positive wild-type (D) and Pptc7 KO (E) MEFs showing high mitophagy rates in response to variable DFO concentrations. (D, E, F) Statistical analysis of data by ordinary one-way ANOVA shown in (D, E), gray bars = wild-type samples, pink bars = Pptc7 KO samples. **** P < 0.0001, *** P < 0.001, ** P < 0.01, ns = not significant. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Western Blot, Plasmid Preparation, Control

    (A) DepMap essentiality profiles of PPTC7 gene effect (x-axis) and FBXL4 gene effect (y-axis). Linear regression analysis and associated P -value shown. (B, C, D, E) Statistical analysis of BNIP3 and NIX monomer and dimer turnover rate data in wild-type (gray bars) and PPTC7 KO (red bards) 293T cells shown in . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) DepMap essentiality profiles of PPTC7 gene effect (x-axis) and FBXL4 gene effect (y-axis). Linear regression analysis and associated P -value shown. (B, C, D, E) Statistical analysis of BNIP3 and NIX monomer and dimer turnover rate data in wild-type (gray bars) and PPTC7 KO (red bards) 293T cells shown in . **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns = not significant, ordinary one-way ANOVA. Error bars represent SD, data points represent individual biological replicates (n = 3).

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques:

    (A) Schematic of DFO treatment and washout timeline and mechanism; figure made with BioRender. (B) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout, when applicable. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (C) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (C, D) Quantification of data shown in (C). BNIP3 monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (E) Western blot of endogenous NIX protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (E, F) Quantification of data shown in (E). NIX monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (G) Western blots of endogenous BNIP3 (top panel) and NIX (bottom panel) in wild-type and PPTC7 KO 293T cells upon treatment with 10 μM MG-132 for the indicated timeframes. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (G, H) Quantification of BNIP3 and NIX monomer (left graphs) and dimer (right graphs) populations in wild-type (gray) and PPTC7 KO (pink) cells shown in (G). Bands were quantified using densitometry, averaged, and plotted over time. Data were analyzed via linear regression, and the significance between slopes was calculated using Analysis of Covariance (ANCOVA). (I) Western blot of endogenous BNIP3 and NIX proteins in wild-type and PPTC7 KO cells after DFO treatment and subsequent washout in the presence or absence of 10 μM MG-132. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control.

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) Schematic of DFO treatment and washout timeline and mechanism; figure made with BioRender. (B) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout, when applicable. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (C) Western blot of endogenous BNIP3 protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (C, D) Quantification of data shown in (C). BNIP3 monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (E) Western blot of endogenous NIX protein after indicated times of DFO treatment and washout in wild-type (left panel) and PPTC7 KO (right panel) 293T cells. “D” indicates dimer species; “M” indicates monomer species. Actin shown as loading control. (E, F) Quantification of data shown in (E). NIX monomer (left graph) or dimer (right graph) bands were quantified using densitometry, averaged, and plotted over time. Data were fit with a one-phase decay model to calculate protein half-lives (T 1/2 ), which are shown below each graph. Error bars represent standard deviations of normalized densitometry across three independent experiments. (G) Western blots of endogenous BNIP3 (top panel) and NIX (bottom panel) in wild-type and PPTC7 KO 293T cells upon treatment with 10 μM MG-132 for the indicated timeframes. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control. (G, H) Quantification of BNIP3 and NIX monomer (left graphs) and dimer (right graphs) populations in wild-type (gray) and PPTC7 KO (pink) cells shown in (G). Bands were quantified using densitometry, averaged, and plotted over time. Data were analyzed via linear regression, and the significance between slopes was calculated using Analysis of Covariance (ANCOVA). (I) Western blot of endogenous BNIP3 and NIX proteins in wild-type and PPTC7 KO cells after DFO treatment and subsequent washout in the presence or absence of 10 μM MG-132. “D” indicates dimer species; “M” indicates monomer species. Actin shown as a loading control.

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Western Blot, Control

    (A) Representative single-plane confocal images of GFP only (top panels) or PPTC7-GFP (bottom panels) expressed in HeLa cells treated for 12 h with 500 μM CoCl 2 . Cells were stained for BNIP3 (second column) or TOMM20 (third column). Overlays are shown for GFP and TOMM20 (fourth column) and GFP and BNIP3 (fifth column). Scale bar = 20 μm. (A, B) Quantification of data shown in (A), mitochondrial BNIP3 staining versus non-specific staining in cells overexpressing GFP only or PPTC7-GFP versus matched untransfected controls for each experiment. Error bars represent the standard error of the mean of three independent experiments. (C) Schematic of PPTC7 features, including a mitochondrial targeting sequence (MTS) and PP2C phosphatase domain, top. Bottom, AlphaFold2 representation of PPTC7 structure; predicted disordered N-terminus and PP2C phosphatase domains indicated. (D) Western blot of 293T cells overexpressing PPTC7 or a ΔMTS-PPTC7 mutant. Red arrows indicate dual species in wild-type PPTC7 expression. * represents a non-specific band. Actin shown as a loading control. (E) PPTC7 protease protection assay. Mitochondria from HeLa FLP-IN cells expressing PPTC7 were isolated and treated with SDS, proteinase K, or both and resolved via SDS–PAGE. Western blots of PPTC7 (top), the outer mitochondrial membrane marker NIX (middle) and the matrix marker TFAM (bottom) shown. (F, G, H) Western blots of BNIP3, NIX, and PPTC7 depicting the ability of various mutants of PPTC7 to suppress BNIP3 and NIX accumulation in response to CoCl 2 treatment. “D” indicates dimer species, “M” indicates monomer species. In (F), a catalytic mutant of PPTC7, D78A, cannot effectively suppress BNIP3 and NIX accumulation relative to wild-type PPTC7. In (G), the ΔMTS-PPTC7 mutant partially or fully suppresses BNIP3 and NIX accumulation, respectively. In (H), a mutant that artificially anchors PPTC7 to the outer mitochondrial membrane, ΔMTS-PPTC7-OMP25, suppresses BNIP3 and NIX accumulation. Actin shown as a loading control.

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) Representative single-plane confocal images of GFP only (top panels) or PPTC7-GFP (bottom panels) expressed in HeLa cells treated for 12 h with 500 μM CoCl 2 . Cells were stained for BNIP3 (second column) or TOMM20 (third column). Overlays are shown for GFP and TOMM20 (fourth column) and GFP and BNIP3 (fifth column). Scale bar = 20 μm. (A, B) Quantification of data shown in (A), mitochondrial BNIP3 staining versus non-specific staining in cells overexpressing GFP only or PPTC7-GFP versus matched untransfected controls for each experiment. Error bars represent the standard error of the mean of three independent experiments. (C) Schematic of PPTC7 features, including a mitochondrial targeting sequence (MTS) and PP2C phosphatase domain, top. Bottom, AlphaFold2 representation of PPTC7 structure; predicted disordered N-terminus and PP2C phosphatase domains indicated. (D) Western blot of 293T cells overexpressing PPTC7 or a ΔMTS-PPTC7 mutant. Red arrows indicate dual species in wild-type PPTC7 expression. * represents a non-specific band. Actin shown as a loading control. (E) PPTC7 protease protection assay. Mitochondria from HeLa FLP-IN cells expressing PPTC7 were isolated and treated with SDS, proteinase K, or both and resolved via SDS–PAGE. Western blots of PPTC7 (top), the outer mitochondrial membrane marker NIX (middle) and the matrix marker TFAM (bottom) shown. (F, G, H) Western blots of BNIP3, NIX, and PPTC7 depicting the ability of various mutants of PPTC7 to suppress BNIP3 and NIX accumulation in response to CoCl 2 treatment. “D” indicates dimer species, “M” indicates monomer species. In (F), a catalytic mutant of PPTC7, D78A, cannot effectively suppress BNIP3 and NIX accumulation relative to wild-type PPTC7. In (G), the ΔMTS-PPTC7 mutant partially or fully suppresses BNIP3 and NIX accumulation, respectively. In (H), a mutant that artificially anchors PPTC7 to the outer mitochondrial membrane, ΔMTS-PPTC7-OMP25, suppresses BNIP3 and NIX accumulation. Actin shown as a loading control.

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Staining, Sequencing, Western Blot, Mutagenesis, Expressing, Control, Isolation, SDS Page, Membrane, Marker

    (A) FACS data shown for data represented in . Cells undergoing high mitophagy are above the diagonal line; percentages indicated in the figure. (B) Quantification of mt-Keima positive mitophagic flux in HeLa FLP-IN TREx cells expressing vector only (left) or PPTC7-FLAG in the presence of 10 μM doxycycline (dox, to promote PPTC7 expression), 200 μM cobalt chloride (CoCl 2 ), or both. ** P < 0.01, ns = not significant, ordinary one-way ANOVA. Error bars represent SD. Each dot represents an independent biological replicate (n = 3). (C) Western blot of PPTC7 expressed in wild-type MEFs (black arrows), Pptc7 KO MEFs, or Pptc7 KO MEFs rescued with human PPTC7 (red arrows). * represents a non-specific band. Basal BNIP3 levels across samples shown below; actin shown as a loading control. (D) Western blot of BNIP3 in crude mitochondria isolated from wild-type or PPTC7 KO 293T cells. Mitochondria were left untreated, treated with recombinant PPTC7, or treated with recombinant PPTC7 D78A. Revert stain is shown for loading; equal loading of recombinant proteins can be seen as depicted by arrows. (E) Alphafold2 model of PPTC7-NIX interaction suggesting PPTC7 D78 and NIX S146 as binding sites. (F) Western blot of NIX monomer immunoprecipitation with FLAG-tagged PPTC7 WT and PPTC7 D78A in whole cell extract. Whole-cell extracts were subjected to immunoprecipitation with anti-FLAG agarose beads and immunoblotted as indicated. Revert stain shown as a loading control.

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) FACS data shown for data represented in . Cells undergoing high mitophagy are above the diagonal line; percentages indicated in the figure. (B) Quantification of mt-Keima positive mitophagic flux in HeLa FLP-IN TREx cells expressing vector only (left) or PPTC7-FLAG in the presence of 10 μM doxycycline (dox, to promote PPTC7 expression), 200 μM cobalt chloride (CoCl 2 ), or both. ** P < 0.01, ns = not significant, ordinary one-way ANOVA. Error bars represent SD. Each dot represents an independent biological replicate (n = 3). (C) Western blot of PPTC7 expressed in wild-type MEFs (black arrows), Pptc7 KO MEFs, or Pptc7 KO MEFs rescued with human PPTC7 (red arrows). * represents a non-specific band. Basal BNIP3 levels across samples shown below; actin shown as a loading control. (D) Western blot of BNIP3 in crude mitochondria isolated from wild-type or PPTC7 KO 293T cells. Mitochondria were left untreated, treated with recombinant PPTC7, or treated with recombinant PPTC7 D78A. Revert stain is shown for loading; equal loading of recombinant proteins can be seen as depicted by arrows. (E) Alphafold2 model of PPTC7-NIX interaction suggesting PPTC7 D78 and NIX S146 as binding sites. (F) Western blot of NIX monomer immunoprecipitation with FLAG-tagged PPTC7 WT and PPTC7 D78A in whole cell extract. Whole-cell extracts were subjected to immunoprecipitation with anti-FLAG agarose beads and immunoblotted as indicated. Revert stain shown as a loading control.

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Isolation, Recombinant, Staining, Binding Assay, Immunoprecipitation

    (A) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with or without 24-h deferoxamine (DFO) treatment. PPTC7-V5-miniTurbo, as well as vector only or V5-miniTurbo-only controls, were transfected into 293T cells. Streptavidin pulldowns were used to enrich for PPTC7-V5-miniTurbo interactors, which were run on SDS–PAGE gels and western blotted for BNIP3 (top blot) or NIX (second blot). Only PPTC7-V5-miniTurbo + biotin samples pulled down BNIP3 and NIX (lanes 6 and 12, streptavidin pull-down gels), indicating specific binding. Western blots shown for reaction input for pulldowns for V5 (showing miniTurbo constructs), BNIP3, NIX, and actin (serving as a load control). (B) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with after 24 h DFO treatment with or without 4 h DFO washout. Streptavidin pulldowns were used to enrich PPTC7-V5-miniTurbo interactors as described in (A). Western blots shown for reaction input for pulldowns as described in (A). (C) A representative maximum z-projection confocal image (left) and corresponding single plane insets (right) are shown of a U2OS cell overexpressing PPTC7-GFP and treated with deferiprone for 24 h. Cells were fixed and stained for BNIP3 and TOMM20 to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 449). (D) As in (C) for cells treated for 24 h with deferiprone and washed for an additional 4 h before fixation. Cells were stained to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 526).

    Journal: Life Science Alliance

    Article Title: Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX

    doi: 10.26508/lsa.202402765

    Figure Lengend Snippet: (A) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with or without 24-h deferoxamine (DFO) treatment. PPTC7-V5-miniTurbo, as well as vector only or V5-miniTurbo-only controls, were transfected into 293T cells. Streptavidin pulldowns were used to enrich for PPTC7-V5-miniTurbo interactors, which were run on SDS–PAGE gels and western blotted for BNIP3 (top blot) or NIX (second blot). Only PPTC7-V5-miniTurbo + biotin samples pulled down BNIP3 and NIX (lanes 6 and 12, streptavidin pull-down gels), indicating specific binding. Western blots shown for reaction input for pulldowns for V5 (showing miniTurbo constructs), BNIP3, NIX, and actin (serving as a load control). (B) Proximity labeling of PPTC7-V5-miniTurbo in 293T cells with after 24 h DFO treatment with or without 4 h DFO washout. Streptavidin pulldowns were used to enrich PPTC7-V5-miniTurbo interactors as described in (A). Western blots shown for reaction input for pulldowns as described in (A). (C) A representative maximum z-projection confocal image (left) and corresponding single plane insets (right) are shown of a U2OS cell overexpressing PPTC7-GFP and treated with deferiprone for 24 h. Cells were fixed and stained for BNIP3 and TOMM20 to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 449). (D) As in (C) for cells treated for 24 h with deferiprone and washed for an additional 4 h before fixation. Cells were stained to visualize co-enrichment of PPTC7 with BNIP3-enriched foci (n = 526).

    Article Snippet: Primary antibodies used in immunoblotting include: anti-human BNIP3 (catalog #44060, dilution 1:1,000, 48 h incubation at 4°C; Cell Signaling Technology [CST]), anti-rodent BNIP3 (catalog #3769, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-NIX (catalog #12396, dilution 1:1,000, 48 h incubation at 4°C; CST), anti-PPTC7 (catalog #NBP190654, dilution 1:1,000, 48 h incubation at 4°C; Novus), anti-HIF-1α (catalog #36169, dilution 1:1,000, overnight incubation at 4°C; CST), anti-β-actin (catalog #3700, dilution 1:1,000; CST; catalog #4970, dilution 1:1,000; CST; and catalog #ab170325, dilution 1:1,000; overnight incubation at 4°C; Abcam), anti-FLAG (catalog #F1804, dilution 1:2,000, overnight incubation at 4°C; Sigma-Aldrich), anti-V5 (catalog #PIMA515253, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific), and anti-myc (catalog #MA121316, dilution 1:1,000, overnight incubation at 4°C; Thermo Fisher Scientific).

    Techniques: Labeling, Plasmid Preparation, Transfection, SDS Page, Western Blot, Binding Assay, Construct, Control, Staining

    Figure 3. BNIP3 enhances mt-reactive oxygen species production, and attenuates mt-membrane

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction.

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Figure 3. BNIP3 enhances mt-reactive oxygen species production, and attenuates mt-membrane

    Article Snippet: The following primary antibodies were used: total OXPHOS rodent antibody cocktail, HADHA, HADHB, MCAD, VDAC1, SLC25A4, SLC25A5, SLC25A11 (Abcam, Boston, MA, USA); BNIP3 rodent specific, GAPDH, AMPKα, p-S485/491-AMPKα, p-T197-AMPKα, GSK3α, p-S21-GSK3α, GSK3β, p-S9-GSK3β, TNNI3, p-S23/24-TNNI3, and PP1a (Cell signaling, Danvers, MA, USA); PGC1α (Novus Biologicals, Centennial, CO, USA); LETM1 (Proteintech, Rosemont, IL, USA); SERCA2A, PLN, and p-S16-PLN (Badrilla, Leeds, UK).

    Techniques: Membrane

    Figure 7. BNIP3 interactome in human and rat LV myocardia identified via BNIP3 co- immunoprecipitation (Co-IP) and mass spectrometry. (A). Venn diagrams show the total number of identified BNIP3 interacting proteins by mass spectrometry in the human (red) and rat (green) HFrEF LV myocardium. The intersection between the two Venn diagrams (black circle) shows the number of commonly identified BNIP3 interacting proteins in human and rat samples. (B,C). Heat maps and PCA plots show the relative log2-fold expression and the variance in biological samples, respectively, in Sham and HFrEF in the rat LV myocardium of the 516 common identified BNIP3 interacting proteins (left), including those that were differentially expressed in HFrEF vs. Sham, taking a cutoff p-value of < 0.05 (right). (D). Heat maps show the relative log2-fold expression in Sham and HFrEF in the rat LV myocardium of some of the important identified BNIP3 interacting proteins that were commonly identified in rat and human HFrEF samples, presented by HFrEF vs. Sham cutoff p-value < 0.05 (left), 0.05 < p < 0.1 (middle), and p > 0.1 (right). (E). Western blot showing the expression of sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) and the mt-proton/calcium exchanger protein (LETM1) in Sham, ShLuc, and ShBNIP3, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc; m, monomer; t, trimer. (F,G). Heat maps of the top Canonical Pathways and Upstream Regulators that were

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction.

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Figure 7. BNIP3 interactome in human and rat LV myocardia identified via BNIP3 co- immunoprecipitation (Co-IP) and mass spectrometry. (A). Venn diagrams show the total number of identified BNIP3 interacting proteins by mass spectrometry in the human (red) and rat (green) HFrEF LV myocardium. The intersection between the two Venn diagrams (black circle) shows the number of commonly identified BNIP3 interacting proteins in human and rat samples. (B,C). Heat maps and PCA plots show the relative log2-fold expression and the variance in biological samples, respectively, in Sham and HFrEF in the rat LV myocardium of the 516 common identified BNIP3 interacting proteins (left), including those that were differentially expressed in HFrEF vs. Sham, taking a cutoff p-value of < 0.05 (right). (D). Heat maps show the relative log2-fold expression in Sham and HFrEF in the rat LV myocardium of some of the important identified BNIP3 interacting proteins that were commonly identified in rat and human HFrEF samples, presented by HFrEF vs. Sham cutoff p-value < 0.05 (left), 0.05 < p < 0.1 (middle), and p > 0.1 (right). (E). Western blot showing the expression of sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) and the mt-proton/calcium exchanger protein (LETM1) in Sham, ShLuc, and ShBNIP3, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc; m, monomer; t, trimer. (F,G). Heat maps of the top Canonical Pathways and Upstream Regulators that were

    Article Snippet: The following primary antibodies were used: total OXPHOS rodent antibody cocktail, HADHA, HADHB, MCAD, VDAC1, SLC25A4, SLC25A5, SLC25A11 (Abcam, Boston, MA, USA); BNIP3 rodent specific, GAPDH, AMPKα, p-S485/491-AMPKα, p-T197-AMPKα, GSK3α, p-S21-GSK3α, GSK3β, p-S9-GSK3β, TNNI3, p-S23/24-TNNI3, and PP1a (Cell signaling, Danvers, MA, USA); PGC1α (Novus Biologicals, Centennial, CO, USA); LETM1 (Proteintech, Rosemont, IL, USA); SERCA2A, PLN, and p-S16-PLN (Badrilla, Leeds, UK).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

    Figure 8. Schematic drawings highlight some of the key findings of the effect of BNIP3 knockdown in the rat pressure overload HFrEF model. These are presented as ShL vs. Sham (A) and ShB vs. ShL (B). The green and red color intensities show the degree of downregulation and upregulation of activity, respectively, or the log2-fold change in protein or phosphosite expression, as noted by the scale bar at the bottom right side of the schematic drawing. The double-headed arrows show the interaction between BNIP3 and its interacting protein. The blue and orange colors show whether there was inhibition/downregulation or activation/upregulation as a result of this interaction. The straight black arrows point to an effect of BNIP3 on protein phosphorylation. The black asterisk denotes

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction.

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Figure 8. Schematic drawings highlight some of the key findings of the effect of BNIP3 knockdown in the rat pressure overload HFrEF model. These are presented as ShL vs. Sham (A) and ShB vs. ShL (B). The green and red color intensities show the degree of downregulation and upregulation of activity, respectively, or the log2-fold change in protein or phosphosite expression, as noted by the scale bar at the bottom right side of the schematic drawing. The double-headed arrows show the interaction between BNIP3 and its interacting protein. The blue and orange colors show whether there was inhibition/downregulation or activation/upregulation as a result of this interaction. The straight black arrows point to an effect of BNIP3 on protein phosphorylation. The black asterisk denotes

    Article Snippet: The following primary antibodies were used: total OXPHOS rodent antibody cocktail, HADHA, HADHB, MCAD, VDAC1, SLC25A4, SLC25A5, SLC25A11 (Abcam, Boston, MA, USA); BNIP3 rodent specific, GAPDH, AMPKα, p-S485/491-AMPKα, p-T197-AMPKα, GSK3α, p-S21-GSK3α, GSK3β, p-S9-GSK3β, TNNI3, p-S23/24-TNNI3, and PP1a (Cell signaling, Danvers, MA, USA); PGC1α (Novus Biologicals, Centennial, CO, USA); LETM1 (Proteintech, Rosemont, IL, USA); SERCA2A, PLN, and p-S16-PLN (Badrilla, Leeds, UK).

    Techniques: Knockdown, Activity Assay, Phospho-proteomics, Expressing, Inhibition, Activation Assay

    Validation of the LV and RV proteomic findings. ( A ). “Core Analyses” was performed in IPA for each of the two-group comparisons, i.e., ShLuc vs. Sham and ShBNIP3 vs. ShLuc, with a cutoff p -value < 0.05. Analyzed datasets were then compared with each other in IPA’s “Comparison Analyses” function to yield the most enriched Canonical Metabolic Pathways (left) and Upstream Regulators (right) that were shared among the two-group comparisons. The respective z-score-based heat maps indicate shared Canonical Pathways and Upstream Regulators that were upregulated/activated or downregulated/inhibited in the two-group comparisons, with orange and blue color intensities representing the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. ( B ). Oxidative phosphorylation (OXPHOS) network of the electron transport chain (ETC) complexes I–V transport system presented in ShLuc vs. Sham (upper panel) and ShBNIP3 vs. ShLuc (lower panel) in LV (left) and RV (right) proteomic datasets. The network highlights mt-OXPHOS proteins in each ETC complex that were downregulated (green) or upregulated (red) or were unchanged (grey) within the two-group comparisons, and those that were not identified (white) in the LV and RV proteomic datasets. The darker the green or red color, the higher the degree of decrease or increase in relative expression, respectively. ( C , D ). Expression of the ETC complexes I–V, BNIP3, and molecular markers involved in mt-biogenesis and metabolism by western blot, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. Abbreviations: ShL, ShLuc; ShB, ShBNIP3.

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Validation of the LV and RV proteomic findings. ( A ). “Core Analyses” was performed in IPA for each of the two-group comparisons, i.e., ShLuc vs. Sham and ShBNIP3 vs. ShLuc, with a cutoff p -value < 0.05. Analyzed datasets were then compared with each other in IPA’s “Comparison Analyses” function to yield the most enriched Canonical Metabolic Pathways (left) and Upstream Regulators (right) that were shared among the two-group comparisons. The respective z-score-based heat maps indicate shared Canonical Pathways and Upstream Regulators that were upregulated/activated or downregulated/inhibited in the two-group comparisons, with orange and blue color intensities representing the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. ( B ). Oxidative phosphorylation (OXPHOS) network of the electron transport chain (ETC) complexes I–V transport system presented in ShLuc vs. Sham (upper panel) and ShBNIP3 vs. ShLuc (lower panel) in LV (left) and RV (right) proteomic datasets. The network highlights mt-OXPHOS proteins in each ETC complex that were downregulated (green) or upregulated (red) or were unchanged (grey) within the two-group comparisons, and those that were not identified (white) in the LV and RV proteomic datasets. The darker the green or red color, the higher the degree of decrease or increase in relative expression, respectively. ( C , D ). Expression of the ETC complexes I–V, BNIP3, and molecular markers involved in mt-biogenesis and metabolism by western blot, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. Abbreviations: ShL, ShLuc; ShB, ShBNIP3.

    Article Snippet: The following primary antibodies were used for the western blot part of the experiment: SERCA2A (Badrilla, Leeds, UK); BNIP3 rodent specific and BNIP3 human specific (Cell signaling, Danvers, MA, USA); LETM1 (Proteintech, Rosemont, IL, USA).

    Techniques: Biomarker Discovery, Comparison, Activation Assay, Inhibition, Phospho-proteomics, Expressing, Western Blot

    BNIP3 enhances mt-reactive oxygen species production, and attenuates mt-membrane potential and respiration, in cultured adult cardiomyocytes in vitro. ( A , B ). Representative fluorescent photomicrographs in cultured adult cardiac myocytes (ACM) transfected with adenovirus encoding for Null, BNIP3, or BNIP3 ShRNA (ShBNIP3), and loaded with the fluorophores TMRM and MitoSOX to assess mt-membrane potential and mt-reactive oxygen species, respectively. Live imaging was acquired at baseline and after 2 and 12 h of phenylephrine (PE, 10 μM) stress. Images are 20× magnified, scale bar 50 μm. Data were obtained from at least 3 independent experiments, and are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( C , D ). Western blots show BNIP3 expression in normal ( C ) and in PE-stressed ( D ) ACM transfected with adenovirus Null (N), BNIP3 (B) or ShBNIP3 (ShB), * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ). Representative tracings of mt-respiration by Oroboros oxygraph-2k system in permeabilized ACM transfected with adenovirus Null, BNIP3, or ShBNIP3. Data were obtained from 7 independent experiments and are presented as mean ± SD, * p < 0.05, ** p < 0.01, **** p < 0.0001. Abbreviations: F.I., fluorescence intensity; ns, not significant; Glu/Mal, glutamate/malate; ADP, adenosine diphosphate; Succ, succinate; Rot, rotenone; Oligo, oligomycin; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; AA, Antimycin A; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride.

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: BNIP3 enhances mt-reactive oxygen species production, and attenuates mt-membrane potential and respiration, in cultured adult cardiomyocytes in vitro. ( A , B ). Representative fluorescent photomicrographs in cultured adult cardiac myocytes (ACM) transfected with adenovirus encoding for Null, BNIP3, or BNIP3 ShRNA (ShBNIP3), and loaded with the fluorophores TMRM and MitoSOX to assess mt-membrane potential and mt-reactive oxygen species, respectively. Live imaging was acquired at baseline and after 2 and 12 h of phenylephrine (PE, 10 μM) stress. Images are 20× magnified, scale bar 50 μm. Data were obtained from at least 3 independent experiments, and are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( C , D ). Western blots show BNIP3 expression in normal ( C ) and in PE-stressed ( D ) ACM transfected with adenovirus Null (N), BNIP3 (B) or ShBNIP3 (ShB), * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ). Representative tracings of mt-respiration by Oroboros oxygraph-2k system in permeabilized ACM transfected with adenovirus Null, BNIP3, or ShBNIP3. Data were obtained from 7 independent experiments and are presented as mean ± SD, * p < 0.05, ** p < 0.01, **** p < 0.0001. Abbreviations: F.I., fluorescence intensity; ns, not significant; Glu/Mal, glutamate/malate; ADP, adenosine diphosphate; Succ, succinate; Rot, rotenone; Oligo, oligomycin; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; AA, Antimycin A; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride.

    Article Snippet: The following primary antibodies were used for the western blot part of the experiment: SERCA2A (Badrilla, Leeds, UK); BNIP3 rodent specific and BNIP3 human specific (Cell signaling, Danvers, MA, USA); LETM1 (Proteintech, Rosemont, IL, USA).

    Techniques: Membrane, Cell Culture, In Vitro, Transfection, shRNA, Imaging, Western Blot, Expressing, Fluorescence

    Validation of the LV and RV phosphoproteomic findings. ( A ). Heat maps of the Canonical signaling pathways that were enriched by −Log 10 p -value in the phosphoproteomic datasets. The orange and blue color intensities represent the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. The heat maps were generated in IPA after “Core Analyses” of each of the two-group comparisons, which were then compared with each other in IPA’s “Comparison Analyses” function. ( B ). Cardiac β-adrenergic signaling network of the phosphoproteins/phosphosites in ShLuc vs. Sham (upper panel) and ShBNIP3 vs. ShLuc (lower panel) in LV (left) and RV (right) phosphoproteomic datasets. The network highlights phosphoproteins/phosphosites that were differentially downregulated (green) or upregulated (red) or were unchanged (grey) in the two-group comparisons, and those that were not identified (white) in the LV and RV phosphoproteomic datasets. The darker the green or red color, the higher the degree of decrease or increase in relative expression, respectively. ( C ). Western blot validating the change in phospholamban (PLN) phosphorylation at serine 16 residue (p-S16-PLN) in Sham, ShLuc, and ShBNIP3 experimental groups, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. ( D – F ). Western blot validating the change in BNIP3 and VDAC1 phosphorylation at serine 79 (p-S79-BNIP3), serine 104 (p-S104-VDAC1) and serine 241 (p-S241-VDAC1) residues, respectively, in Sham, ShLuc, and ShBNIP3 experimental groups, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. Abbreviations: p, pentamer; m, monomer; d, dimer; te, tetramer; he, hexamer.

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Validation of the LV and RV phosphoproteomic findings. ( A ). Heat maps of the Canonical signaling pathways that were enriched by −Log 10 p -value in the phosphoproteomic datasets. The orange and blue color intensities represent the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. The heat maps were generated in IPA after “Core Analyses” of each of the two-group comparisons, which were then compared with each other in IPA’s “Comparison Analyses” function. ( B ). Cardiac β-adrenergic signaling network of the phosphoproteins/phosphosites in ShLuc vs. Sham (upper panel) and ShBNIP3 vs. ShLuc (lower panel) in LV (left) and RV (right) phosphoproteomic datasets. The network highlights phosphoproteins/phosphosites that were differentially downregulated (green) or upregulated (red) or were unchanged (grey) in the two-group comparisons, and those that were not identified (white) in the LV and RV phosphoproteomic datasets. The darker the green or red color, the higher the degree of decrease or increase in relative expression, respectively. ( C ). Western blot validating the change in phospholamban (PLN) phosphorylation at serine 16 residue (p-S16-PLN) in Sham, ShLuc, and ShBNIP3 experimental groups, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. ( D – F ). Western blot validating the change in BNIP3 and VDAC1 phosphorylation at serine 79 (p-S79-BNIP3), serine 104 (p-S104-VDAC1) and serine 241 (p-S241-VDAC1) residues, respectively, in Sham, ShLuc, and ShBNIP3 experimental groups, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc. Abbreviations: p, pentamer; m, monomer; d, dimer; te, tetramer; he, hexamer.

    Article Snippet: The following primary antibodies were used for the western blot part of the experiment: SERCA2A (Badrilla, Leeds, UK); BNIP3 rodent specific and BNIP3 human specific (Cell signaling, Danvers, MA, USA); LETM1 (Proteintech, Rosemont, IL, USA).

    Techniques: Biomarker Discovery, Protein-Protein interactions, Activation Assay, Inhibition, Generated, Comparison, Expressing, Western Blot, Phospho-proteomics, Residue

    BNIP3 interactome in human and rat LV myocardia identified via BNIP3 co-immunoprecipitation (Co-IP) and mass spectrometry. ( A ). Venn diagrams show the total number of identified BNIP3 interacting proteins by mass spectrometry in the human (red) and rat (green) HFrEF LV myocardium. The intersection between the two Venn diagrams (black circle) shows the number of commonly identified BNIP3 interacting proteins in human and rat samples. ( B , C ) . Heat maps and PCA plots show the relative log 2 -fold expression and the variance in biological samples, respectively, in Sham and HFrEF in the rat LV myocardium of the 516 common identified BNIP3 interacting proteins (left), including those that were differentially expressed in HFrEF vs. Sham, taking a cutoff p -value of <0.05 (right). ( D ). Heat maps show the relative log 2 -fold expression in Sham and HFrEF in the rat LV myocardium of some of the important identified BNIP3 interacting proteins that were commonly identified in rat and human HFrEF samples, presented by HFrEF vs. Sham cutoff p -value < 0.05 (left), 0.05 < p < 0.1 (middle), and p > 0.1 (right). ( E ). Western blot showing the expression of sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) and the mt-proton/calcium exchanger protein (LETM1) in Sham, ShLuc, and ShBNIP3, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc; m, monomer; t, trimer. ( F , G ). Heat maps of the top Canonical Pathways and Upstream Regulators that were upregulated/activated or downregulated/inhibited in HFrEF vs. Sham rat samples. The orange and blue color intensities represent the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. The heat maps were generated in IPA after “Core Analyses” of each of the two-group comparisons (HFrEF vs. Sham), taking a cutoff p -value of <0.05 vs. <0.1, which were then compared with each other in IPA’s “Comparison Analyses” function. ( H ). BNIP3 interactome enriched metabolic reactome pathways, by q-value, that were attenuated (blue bars) in HFrEF vs. Sham. Data are presented as percentage of total protein count per reactome pathway. ( I – K ). Validation of some of the BNIP3 interacting proteins by Co-IP and western blot in Normal ACM ( I ), Rat LV myocardium, Sham vs. HFrEF ( J ), and human HFrEF LV myocardium ( K ).

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: BNIP3 interactome in human and rat LV myocardia identified via BNIP3 co-immunoprecipitation (Co-IP) and mass spectrometry. ( A ). Venn diagrams show the total number of identified BNIP3 interacting proteins by mass spectrometry in the human (red) and rat (green) HFrEF LV myocardium. The intersection between the two Venn diagrams (black circle) shows the number of commonly identified BNIP3 interacting proteins in human and rat samples. ( B , C ) . Heat maps and PCA plots show the relative log 2 -fold expression and the variance in biological samples, respectively, in Sham and HFrEF in the rat LV myocardium of the 516 common identified BNIP3 interacting proteins (left), including those that were differentially expressed in HFrEF vs. Sham, taking a cutoff p -value of <0.05 (right). ( D ). Heat maps show the relative log 2 -fold expression in Sham and HFrEF in the rat LV myocardium of some of the important identified BNIP3 interacting proteins that were commonly identified in rat and human HFrEF samples, presented by HFrEF vs. Sham cutoff p -value < 0.05 (left), 0.05 < p < 0.1 (middle), and p > 0.1 (right). ( E ). Western blot showing the expression of sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) and the mt-proton/calcium exchanger protein (LETM1) in Sham, ShLuc, and ShBNIP3, * p < 0.05 vs. Sham and † p < 0.05 vs. ShLuc; m, monomer; t, trimer. ( F , G ). Heat maps of the top Canonical Pathways and Upstream Regulators that were upregulated/activated or downregulated/inhibited in HFrEF vs. Sham rat samples. The orange and blue color intensities represent the z-score-based extent of upregulation/activation or downregulation/inhibition, respectively. The heat maps were generated in IPA after “Core Analyses” of each of the two-group comparisons (HFrEF vs. Sham), taking a cutoff p -value of <0.05 vs. <0.1, which were then compared with each other in IPA’s “Comparison Analyses” function. ( H ). BNIP3 interactome enriched metabolic reactome pathways, by q-value, that were attenuated (blue bars) in HFrEF vs. Sham. Data are presented as percentage of total protein count per reactome pathway. ( I – K ). Validation of some of the BNIP3 interacting proteins by Co-IP and western blot in Normal ACM ( I ), Rat LV myocardium, Sham vs. HFrEF ( J ), and human HFrEF LV myocardium ( K ).

    Article Snippet: The following primary antibodies were used for the western blot part of the experiment: SERCA2A (Badrilla, Leeds, UK); BNIP3 rodent specific and BNIP3 human specific (Cell signaling, Danvers, MA, USA); LETM1 (Proteintech, Rosemont, IL, USA).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot, Activation Assay, Inhibition, Generated, Comparison, Biomarker Discovery

    Schematic drawings highlight some of the key findings of the effect of BNIP3 knockdown in the rat pressure overload HFrEF model. These are presented as ShL vs. Sham ( A ) and ShB vs. ShL ( B ). The green and red color intensities show the degree of downregulation and upregulation of activity, respectively, or the log 2 -fold change in protein or phosphosite expression, as noted by the scale bar at the bottom right side of the schematic drawing. The double-headed arrows show the interaction between BNIP3 and its interacting protein. The blue and orange colors show whether there was inhibition/downregulation or activation/upregulation as a result of this interaction. The straight black arrows point to an effect of BNIP3 on protein phosphorylation. The black asterisk denotes changes in protein expression or phosphorylation by western blot only. Abbreviations: GLUT4, glucose transporter member 4; CD36, platelet glycoprotein 4; NCX1, sodium/calcium exchanger 1; LTCC, voltage-dependent L-type calcium channel; ATP1A1, sodium/potassium-transporting ATPase subunit alpha-1; STIM1, stromal interaction molecule 1; ORA1, calcium release-activated calcium channel protein 1; AMPK, 5′-AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; CPT1 and 2, carnitine O-palmitoyltransferase 1 and 2; SLC25a3, mt-phosphate carrier protein; SLC25a11, mt-2-oxoglutarate/malate carrier protein; SLC25a4, ADP/ATP translocase 1; HADHB, mt-trifunctional enzyme subunit beta; FA, fatty acid; Bckdk, mt-3-methyl-2-oxobutanoate dehydrogenase kinase; BCAA, branched-chain amino acid; Bcat, BCAA aminotransferase; BCKAs, branched-chain α-ketoacids; BCKDHA, mt-2-oxoisovalerate dehydrogenase subunit alpha; GSTK1, glutathione S-transferase kappa 1; TXNRD2, mt-thioredoxin reductase 2; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; VDAC1, voltage-dependent anion channel isoform 1; IP3R, inositol 1,4,5-triphosphate receptor; NCLX, mt-sodium/calcium exchanger; LETM1, mt-proton/calcium exchanger; MCU, mt-calcium uniporter; P4HB, protein disulfide isomerase; HSP90B1, heat shock protein 90 beat member 1; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; PLN, phospholamban; RYR2, ryanodine receptor isoform 2; PRKACA, cAMP-dependent protein kinase catalytic subunit alpha; PRKAR2A, cAMP-dependent protein kinase type II-alpha regulatory subunit; PRKAR2B, cAMP-dependent protein kinase type II-beta regulatory subunit; AKAP1 and 18, A-kinase anchor protein 1 and 18; AC, adenylate cyclase; PP1A, serine/threonine-protein phosphatase PP1-alpha catalytic subunit; PPP1R12A and PPP1R12B, PP1 regulatory subunits 12A and 12B; PDE7B, cAMP-specific 3′,5′-cyclic phosphodiesterase 7B; DRP1, dynamin-related protein 1; OPA1, optic atrophy 1; PRKCE, protein kinase C epsilon type; ETC, electron transport chain; MICOS, mt-contact site and cristae organizing system; MIC19, MICOS complex subunit MIC19; EIFs, eukaryotic translation initiation factors; RPSs, 40S ribosomal proteins; RPLs, 60S ribosomal proteins; LIPE, hormone sensitive lipase; DES, desmin; LMNA, lamin A; VCL, vinculin; TLN1, talin-1; PAK4, serine/threonine protein kinase PAK 4; MYO18A, unconventional myosin-XVIIIa.

    Journal: Cells

    Article Title: Multiomics Approach Reveals an Important Role of BNIP3 in Myocardial Remodeling and the Pathogenesis of Heart Failure with Reduced Ejection Fraction

    doi: 10.3390/cells11091572

    Figure Lengend Snippet: Schematic drawings highlight some of the key findings of the effect of BNIP3 knockdown in the rat pressure overload HFrEF model. These are presented as ShL vs. Sham ( A ) and ShB vs. ShL ( B ). The green and red color intensities show the degree of downregulation and upregulation of activity, respectively, or the log 2 -fold change in protein or phosphosite expression, as noted by the scale bar at the bottom right side of the schematic drawing. The double-headed arrows show the interaction between BNIP3 and its interacting protein. The blue and orange colors show whether there was inhibition/downregulation or activation/upregulation as a result of this interaction. The straight black arrows point to an effect of BNIP3 on protein phosphorylation. The black asterisk denotes changes in protein expression or phosphorylation by western blot only. Abbreviations: GLUT4, glucose transporter member 4; CD36, platelet glycoprotein 4; NCX1, sodium/calcium exchanger 1; LTCC, voltage-dependent L-type calcium channel; ATP1A1, sodium/potassium-transporting ATPase subunit alpha-1; STIM1, stromal interaction molecule 1; ORA1, calcium release-activated calcium channel protein 1; AMPK, 5′-AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; CPT1 and 2, carnitine O-palmitoyltransferase 1 and 2; SLC25a3, mt-phosphate carrier protein; SLC25a11, mt-2-oxoglutarate/malate carrier protein; SLC25a4, ADP/ATP translocase 1; HADHB, mt-trifunctional enzyme subunit beta; FA, fatty acid; Bckdk, mt-3-methyl-2-oxobutanoate dehydrogenase kinase; BCAA, branched-chain amino acid; Bcat, BCAA aminotransferase; BCKAs, branched-chain α-ketoacids; BCKDHA, mt-2-oxoisovalerate dehydrogenase subunit alpha; GSTK1, glutathione S-transferase kappa 1; TXNRD2, mt-thioredoxin reductase 2; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; VDAC1, voltage-dependent anion channel isoform 1; IP3R, inositol 1,4,5-triphosphate receptor; NCLX, mt-sodium/calcium exchanger; LETM1, mt-proton/calcium exchanger; MCU, mt-calcium uniporter; P4HB, protein disulfide isomerase; HSP90B1, heat shock protein 90 beat member 1; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; PLN, phospholamban; RYR2, ryanodine receptor isoform 2; PRKACA, cAMP-dependent protein kinase catalytic subunit alpha; PRKAR2A, cAMP-dependent protein kinase type II-alpha regulatory subunit; PRKAR2B, cAMP-dependent protein kinase type II-beta regulatory subunit; AKAP1 and 18, A-kinase anchor protein 1 and 18; AC, adenylate cyclase; PP1A, serine/threonine-protein phosphatase PP1-alpha catalytic subunit; PPP1R12A and PPP1R12B, PP1 regulatory subunits 12A and 12B; PDE7B, cAMP-specific 3′,5′-cyclic phosphodiesterase 7B; DRP1, dynamin-related protein 1; OPA1, optic atrophy 1; PRKCE, protein kinase C epsilon type; ETC, electron transport chain; MICOS, mt-contact site and cristae organizing system; MIC19, MICOS complex subunit MIC19; EIFs, eukaryotic translation initiation factors; RPSs, 40S ribosomal proteins; RPLs, 60S ribosomal proteins; LIPE, hormone sensitive lipase; DES, desmin; LMNA, lamin A; VCL, vinculin; TLN1, talin-1; PAK4, serine/threonine protein kinase PAK 4; MYO18A, unconventional myosin-XVIIIa.

    Article Snippet: The following primary antibodies were used for the western blot part of the experiment: SERCA2A (Badrilla, Leeds, UK); BNIP3 rodent specific and BNIP3 human specific (Cell signaling, Danvers, MA, USA); LETM1 (Proteintech, Rosemont, IL, USA).

    Techniques: Knockdown, Activity Assay, Phospho-proteomics, Expressing, Inhibition, Activation Assay, Western Blot