Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: SERCA2b is a novel binding partner of TER. A , purification of Strep-TER–binding proteins. The Triton X-100 extracts of Strep-TER–expressing HEK293 cells or parental cells were applied to Strep-Tactin Sepharose. The bound proteins were eluted with desthiobiotin, and subjected to SDS-PAGE followed by silver staining. The bands indicated by arrowheads were cut out and subjected to mass spectrometry analysis. Data are representative of four independent experiments. B , the list of Strep-TER–binding proteins. The identified proteins of the bands in panel A are shown. C , pull-down of endogenous SERCA2b by Strep-TER. The inputs and purified proteins obtained in panel A was subjected to Western blotting with anti-SERCA2b mAb and Strep-Tactin-HRP. The asterisk (∗) indicates a band corresponding to an SDS-resistant heterodimer of SERCA2b and Strep-TER. Data are representative of three independent experiments. D , coimmunoprecipitation of endogenous SERCA2b with endogenous TER. The Triton X-100 extracts of HEK293 cells or the DDM extracts of primary keratinocytes or HuH-7 cells were immunoprecipitated with anti-TER pAb or control Ab and then subjected to Western blotting with anti-TER pAb or anti-SERCA2b mAb. The asterisks (∗) indicate nonspecific bands in the immunoprecipitates (IP). Data are representative of three (HEK293) or two (HuH-7) independent experiments. The experiment using primary keratinocytes was performed once. E , colocalization of SERCA2b and TER. HEK293 cells were fixed and immunostained with anti-TER pAb and anti-SERCA2b mAb. Data are representative of three independent experiments. The magnified view of the boxed area is shown in the right panel. Arrowheads indicate the regions where TER and SERCA2b are colocalized. (Scale bar, 10 μm in the merged image and 3 μm in the magnified image). Pearson’s coefficient between TER and SERCA2b is indicated in the merged image (mean ± SD, n = 28 cells). DDM, n -dodecyl-β-D-maltoside; pAb, polyclonal antibody; SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Binding Assay, Purification, Expressing, SDS Page, Silver Staining, Mass Spectrometry, Western Blot, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: SERCA2b directly binds to TER in a conformation-dependent manner. A , direct binding of SERCA2b to TER in the presence or absence of Ca 2+ . PA-SERCA2b (50 pmol) was incubated with anti-FLAG mAb-conjugated beads with or without immobilized FLAG–TER (125 pmol). The binding reaction was performed in the presence or absence of 100 nM free Ca 2+ . The bound proteins were then eluted with the SDS sample buffer and subjected to SDS-PAGE followed by CBB staining. This experiment was performed once. B , inhibition of the direct binding between SERCA2b and TER by Tg. 0.25 μM PA-SERCA2b was treated with or without 2.5 μM Tg, and the binding experiments were then performed as in panel A in the presence of 100 nM free Ca 2+ . Data are representative of two independent experiments. C , inhibition of the binding between Strep-TER and SERCA2b by Tg. Strep-TER–expressing HEK293 cells or parental cells were treated with or without 1 μM Tg for 90 min. The Triton X-100 extracts of these cells were incubated with Strep-Tactin Sepharose. The bound proteins were then eluted with the SDS sample buffer and subjected to Western blotting with anti-SERCA2b mAb or Strep-Tactin-HRP. Data are representative of four independent experiments. CBB, Coomassie Brilliant Blue; SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase; Tg, thapsigargin.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Binding Assay, Incubation, SDS Page, Staining, Inhibition, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: The C-terminal region of TER is essential for the binding to SERCA2b. A , left , predicted topology of TER. The topology was based on previous literatures ( , ). Right , domain structure of full-length TER and the deletion mutants used in this study. B , coimmunoprecipitation analysis of SERCA2b and TER deletion mutants. 3xHA-SERCA2b and FLAG-empty vector, FLAG–TER FL, FLAG–TER-N-term, FLAG–TER (ΔC or ΔN), or FLAG–KAR were transfected into HEK293 cells. KAR, another essential component of the FA elongation cycle, was included as a negative control. The Triton X-100 extracts of the transfected cells were immunoprecipitated with anti-FLAG mAb and then subjected to Western blotting with anti-HA mAb or anti-FLAG pAb. The asterisks (∗) indicate nonspecific bands in the IP. Data are representative of three independent experiments. C , binding of the C-terminal region of TER to SERCA2b. 3xHA-SERCA2b was transfected into HEK293 cells, and the Triton X-100 extracts of the transfected cells were incubated with 2.8 nmol of GST, GST-TER-N-term, or GST-TER-C-term immobilized on glutathione Sepharose. The bound proteins were eluted with the SDS sample buffer and subjected to SDS-PAGE followed by Western blotting with anti-HA mAb or CBB staining. Data are representative of two independent experiments. D , inhibition of the binding between the C-terminal region of TER and SERCA2b by Tg. HEK293 cells expressing 3xHA-SERCA2b were treated with or without 1 μM Tg for 90 min, and the Triton X-100 extracts of these cells were subjected to the pull-down assay as in panel C with 1.9 nmol of GST or GST-TER-C-term immobilized on glutathione Sepharose. This experiment was performed once. The double asterisks (∗∗) in panels C and D indicate the degradation products of GST-TER-C-term. CBB, Coomassie Brilliant Blue; FA, fatty acid; FLAG–TER, recombinant TER with an N-terminal FLAG-tag; GST, glutathione-S-transferase; KAR, 3-ketoacyl-CoA reductase; SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase; Tg, thapsigargin; TM, transmembrane region; pAb, polyclonal antibody.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Binding Assay, Plasmid Preparation, Transfection, Negative Control, Immunoprecipitation, Western Blot, Incubation, SDS Page, Staining, Inhibition, Expressing, Pull Down Assay, Recombinant, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: Reduction of the SERCA2b ATPase activity by TER overexpression. A , protein levels of FLAG-tagged TER and SERCA2b in microsomal fractions. FLAG-empty vector, FLAG–TER FL, or ΔC was transfected into HEK293 cells. The microsomal fractions were then prepared by differential centrifugation. The samples were subjected to Western blotting with anti-FLAG mAb, anti-SERCA2b mAb, or anti-Calnexin mAb. Data are representative of four independent experiments. B , SERCA2b ATPase assay. The SERCA2b-dependent release of inorganic phosphate (P i ) from ATP was measured with 30 μg of the microsomal proteins. Data represent the mean ± SD from four independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 versus FLAG-empty vector, two-tailed t -test. SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Activity Assay, Over Expression, Plasmid Preparation, Transfection, Centrifugation, Western Blot, ATPase Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: TER limits the Ca 2+ content in the ER lumen. A , increase of the ER Ca 2+ content by TER depletion. siControl or siTER (#1 or #2) was transfected into HEK293 cells. After 72 h, the cells were subjected to the following experiments. The whole-cell lysates were subjected to Western blotting with anti-TER pAb, anti-SERCA2b mAb, or anti-β-actin mAb. The cells were loaded with a cytosolic Ca 2+ indicator dye, Fluo-4, and then incubated in a Ca 2+ -free buffer supplemented with 0.5 mM EGTA. The Ca 2+ in the ER lumen was released by adding 1 μM Tg (time = 0 s), and the Fluo-4 fluorescence was measured every 5 s for 10 min. The changes in fluorescence values relative to baseline (ΔF/F 0 ) were plotted as a function of time. The areas under the curves, which were normalized to the siControl values, were shown in a bar graph. Data represent the mean ± SEM in the traces, or the mean ± SD with data points plotted in the bar graph, from four independent experiments. B , rescue of the siTER #1 effects by reconstituting TER expression. The indicated siRNA and plasmid were transfected into HEK293 cells. After 72 h, the cells were subjected to the subsequent experiments as in panel A . The whole-cell lysates were subjected to Western blotting with anti-TER pAb, anti-FLAG mAb, or anti-β-actin mAb. Data represent the mean ± SEM in the traces, or the mean ± SD with data points plotted in the bar graph, from three independent experiments. A and B , ∗ p < 0.05 and ∗∗ p < 0.01 versus the theoretical control value of 1.0, two-tailed one-sample t -test. C , direct measurement of the ER Ca 2+ levels with a genetically encoded Ca 2+ indicator. At 48 h after siRNA transfection, HEK293 cells were transfected with the indicator (G-CEPIA1 er ) and further incubated for 24 h. The cells were then incubated in a Ca 2+ -free buffer supplemented with 0.5 mM EGTA and treated with 1 μM Tg (time = 0 s). The G-CEPIA1 er fluorescence was measured every 10 s for 15 min. The fluorescence values relative to the final fluorescence values (F/F final ) were plotted as a function of time. The relative decrease of the G-CEPIA1 er fluorescence values (ΔF/F final ) was shown in a bar graph. Data represent the mean ± SEM in the traces, or the mean ± SD with data points plotted in the bar graph, from five independent experiments. ∗ p < 0.05 versus siControl, two-tailed paired t -test. ER, endoplasmic reticulum; n.s., nonspecific bands; SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase; Tg, thapsigargin.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Transfection, Western Blot, Incubation, Fluorescence, Expressing, Plasmid Preparation, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Trans -2-enoyl-CoA reductase limits Ca 2+ accumulation in the endoplasmic reticulum by inhibiting the Ca 2+ pump SERCA2b

doi: 10.1016/j.jbc.2021.100310

Figure Lengend Snippet: TER depletion shortens the duration of cytosolic Ca 2+ responses in a SERCA2b-dependent manner. A , Ca 2+ responses elicited by ATP. siControl or siTER (#1 or #2) was transfected into HEK293 cells. After 48 h, the cells were loaded with Fluo-4 and then incubated in Ca 2+ -containing buffer. After 30 min, 100 μM ATP was added (time = 0 s), and the Fluo-4 fluorescence was measured every 2 s for 5 min. The changes in fluorescence values relative to baseline (ΔF/F 0 ) were plotted as a function of time in the left graph. To visualize the kinetics of the Ca 2+ responses, the ΔF/F 0 values were normalized to the maximum value in each trace and are shown in the middle graph. Decay time constants of the Ca 2+ traces, which were determined by single exponential curve fitting, are shown in a bar graph in the right . Data represent the mean ± SEM in the traces, or the mean ± SD with data points plotted in the bar graph, from five independent experiments. ∗∗ p < 0.01 versus siControl, two-tailed t -test. B , Ca 2+ responses elicited by ATP in the presence of Tg. Cells were treated as in panel A , except that ATP was added with 1 μM Tg and the fluorescence was measured for 10 min. Data represent the mean ± SEM in the traces, or the mean ± SD with data points plotted in the bar graph, from five independent experiments. SERCA2b, sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b; TER, trans -2-enoyl-CoA reductase; Tg, thapsigargin.

Article Snippet: The following reagents were purchased from the indicated manufacturers: mouse anti-SERCA2b mAb (clone IID8 , Cat. No. sc-53010, for Western blotting) from Santa Cruz Biotechnology; mouse anti-SERCA2b mAb (clone 2A7-A1 , Cat. No. NB300-581, for immunostaining) from Novus; rabbit anti-TER polyclonal antibody (pAb) (Cat. No. A305-515A) from Bethyl Laboratories; horseradish peroxidase–conjugated Strep-Tactin from IBA Lifesciences; rat anti-HA mAb (clone 3F10, Cat. No. 11867431001) from Roche; mouse anti-HA mAb (clone 16B12, Cat. No. 901513) from BioLegend; rabbit anti-FLAG pAb (Cat. No. F7425), mouse anti-FLAG mAb (clone M2, Cat. No. F1804), mouse anti-β-actin mAb (clone AC-74, Cat. No. A2228) from Sigma; rabbit anti-Calnexin mAb (clone C5C9, Cat. No. 2679S) and normal rabbit IgG (Cat. No. 2729S) from Cell Signaling Technology.

Techniques: Transfection, Incubation, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

doi: 10.1038/s41467-020-18607-1

Figure Lengend Snippet: a Superdex 75 gel filtration chromatograms of human NTD and heme. NTD was incubated with heme and run on Superdex 75 gel filtration column. The flow through was measured for absorbance at 230 nm (gray), 380 nm (blue), and 415 nm (green). Absorbance intensity was normalized to maximum value. b Changes in absorption spectrum intensity of heme incubated with different concentrations of wild-type (red) or mutant (gray) NTD (see color scale). Heme (100 µM) absorption was set to zero. c Changes in absorption spectrum intensity of heme (100 µM) incubated with different concentrations of wildtype (blue) or mutant (gray) HP motif peptide (see color scale). Wildtype (WT) and mutant (Mut) peptide sequences are shown. d Co-IP and immunoblotting analysis of HA-tagged MFSD7C and FLAG-tagged CYC1, NDUFA4, COX4I1, ATP5h, ATP5c1, or HMOX1. Shown are representative data from five separate experiments. e Co-IP and immunoblotting analysis of endogenous MFSD7C with ATP5h, SERCA2b and HMOX1 in bone marrow-derived macrophages from Mfsd7c fl/fl or Mfsd7c −/− C57BL/6 mice (see “Methods” section for details). Shown are immunoblots of MFSD7C, ATP5h, SERCA2b, and HMOX1 on whole lysates or immunoblots of MFSD7C on anti-ATP5h, anti-SERCA2b and anti-HMOX1 immunoprecipitates. Representative data from one from three experiments are shown. f Mouse whole brain extract was fractionated using differential centrifugation to enrich for mitochondria and analyzed by immunoblotting against the indicated proteins. Shown are representative data from three separate experiments. WCE: whole cell extract, Sup: supernatant, Mito: 10,000× g mitochondrial fraction. g Immunofluorescent localization of MFSD7C in mitochondria. THP-1 cells were stained with MitoTracker (green), fixed and permeabilized, and then stained with rabbit polyclonal antibody specific for the C-terminus of MFSD7C, followed with Alexa Fluor® 594-labeled goat anti-rabbit antibody (red). Nuclei were labeled using DAPI (blue). Co-localization between MFSD7C and MitoTracker appears as yellow in the merged images. Scale bar in d and f : 10 µm.

Article Snippet: Antibodies specific for SERCA2b (Catalog No. 4388) for Western blotting were purchased from Cell Signaling Technology.

Techniques: Filtration, Incubation, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Centrifugation, Staining, Labeling

Journal: Nature Communications

Article Title: MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

doi: 10.1038/s41467-020-18607-1

Figure Lengend Snippet: List of proteins co-immunoprecipitated with MFSD7C.

Article Snippet: Antibodies specific for SERCA2b (Catalog No. 4388) for Western blotting were purchased from Cell Signaling Technology.

Techniques: Clinical Proteomics, Membrane, Modification

Journal: Nature Communications

Article Title: MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

doi: 10.1038/s41467-020-18607-1

Figure Lengend Snippet: a 293FT cells were transfected with HA-tagged murine MFSD7C and FLAG-tagged murine SERCA2b. Thirty hours later, MG132 was added into half of the cells and the other half was not treated. Another 35 h later, some cells were treated with 40 μM heme for 1 h before lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody, and eluted, followed by anti-HA immunoprecipitation and elution. Total cell lysate and elute were subjected to Western blotting and probed with anti-SERCA2b or anti-MFSD7C antibodies. Shown are representative data from one of three experiments. b 293T cells were co-transfected with HA-MFSD7C and FLAG-SERCA2b. Twenty-four hours later, cells were either not treated or treated with 10 μM MG132 for either 6 or 12 h. The cells were lysed and subjected to FLAG pull-down and blotted with anti-MFSD7C, anti-SERCA2b and anti-ubiquitin antibodies. Shown are representative data from one of three experiments. c SERCA2b protein levels in parental THP-1 cells, 7CKO cells (3D12, A11, B11, and 4B8) or SERCA2b −/− (#1 and #3) cells. Parental THP-1 cells were either not treated or treated with heme before lysis. Shown are representative data from one of four experiments. d THP-1, 4B8, 4B8 FL , and 4B8 ΔN cells were either not treated or treated with 40 μM heme for 1 h, lysed and subjected to Western blotting with anti-MFSD7C (top), anti-SERCA2b (middle), and anti-®-tubulin (bottom). Shown are representative data from one of two experiments. e THP-1 cells were incubated with FPT for 6 h, washed, treated with or without 4 μM thapsigargin for 2 h. Cells were washed and treated with 40 μM heme for 1 h before flow cytometry. Representative FPT histograms from one of three experiments are shown. f Parental and Serca2b −/− THP-1 cells were incubated with FPT for 6 h, washed and treated with or without 40 μM heme for 1 h, followed by flow cytometry. Shown are representative FPT histograms from one of three experiments.

Article Snippet: Antibodies specific for SERCA2b (Catalog No. 4388) for Western blotting were purchased from Cell Signaling Technology.

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Incubation, Flow Cytometry

Journal: Nature Communications

Article Title: MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

doi: 10.1038/s41467-020-18607-1

Figure Lengend Snippet: a , b Proposed models of MFSD7C regulation of mitochondrial respiration in response to heme, with MFSD7C residing in the inner ( a ) or the outer ( b ) mitochondrial membrane. When heme level is low, MFSD7C interacts with ETC components and SERCA2b, leading to SERCA2b ubiquitination and degradation and coupled mitochondrial respiration: increased ATP synthesis and reduced thermogenesis. When heme level is high, heme binding to the NTD of MFSD7C disrupts its interactions with ETC components and SERCA2b, leading to SERCA2b stabilization and uncoupled mitochondrial respiration: increased thermogenesis and reduced ATP synthesis.

Article Snippet: Antibodies specific for SERCA2b (Catalog No. 4388) for Western blotting were purchased from Cell Signaling Technology.

Techniques: Membrane, Ubiquitin Proteomics, Binding Assay