Journal: Frontiers in Marine Science

Article Title: Hypothermal Effects on Energy Supply for Ionocytes in Gills of Freshwater- and Seawater-Acclimated Milkfish, Chanos chanos

doi: 10.3389/fmars.2022.880103

Figure Lengend Snippet: FIGURE 1 | Immunofluorescence staining of gill filaments of milkfish stained with anti-cytochrome c oxidase (COX IV, green; (A, D), citrate synthase (CS, red; (B), sodium-potassium ATPase a1 subunit (NKAa1, red; (E), glycogen phosphorylase (GP, red; (G), sodium-potassium ATPase a subunit (NKA, green; (G, H), and glucose transporter 1 (GLUT1, red; (H) antibodies. Immunoreactions for the merged images (C) and (F) are shown from COX IV (A) with CS (B), and COX IV (D) with NKAa1 (E), respectively. F, filament; L, lamella. Scale bar, 50 mm.

Article Snippet: The following primary antibodies were used in this study: (1) Na+, K+-ATPase a1 subunit (a6F, DSHB, Iowa City, IA, USA); (2) Na+, K+-ATPase a subunit (a5, DSHB, Iowa City, IA, USA); (3) citrate synthase (sc-390693, Santa Cruz Biotechnology, Dallas, TX, USA); (4) COX IV (#4850, Cell Signaling, Beverly, MA, USA); (5) b-actin (GTX109639, Genetex, Irvine, CA, USA); (6) glycogen phosphorylase (GTX124390, Genetex); and (7) glucose transporter 1 (ab128033, Abcam; Figure S1).

Techniques: Staining

Journal: Frontiers in Marine Science

Article Title: Hypothermal Effects on Energy Supply for Ionocytes in Gills of Freshwater- and Seawater-Acclimated Milkfish, Chanos chanos

doi: 10.3389/fmars.2022.880103

Figure Lengend Snippet: FIGURE 4 | Relative protein abundance of branchial citrate synthase (CS) and cytochrome c oxidase IV (COX IV) in freshwater (FW)- and seawater (SW)-acclimated milkfish at 28°C (white bar) and 18°C (striped bar). Representative immunoblots of CS (A) and COX IV (B) show a single immunoreactive band. The single immunoreactive band of b-actin at 42 kDa was used as the loading control. Different letters in the FW milkfish (a, b) and SW milkfish (a’, b’) indicate significant differences between the normal and low-temperature groups. The x and y indicate significant differences between the FW and SW groups at 28°C or 18°C. Values represent the mean ± standard error of the mean (SEM), n = 9, P < 0.05.

Article Snippet: The following primary antibodies were used in this study: (1) Na+, K+-ATPase a1 subunit (a6F, DSHB, Iowa City, IA, USA); (2) Na+, K+-ATPase a subunit (a5, DSHB, Iowa City, IA, USA); (3) citrate synthase (sc-390693, Santa Cruz Biotechnology, Dallas, TX, USA); (4) COX IV (#4850, Cell Signaling, Beverly, MA, USA); (5) b-actin (GTX109639, Genetex, Irvine, CA, USA); (6) glycogen phosphorylase (GTX124390, Genetex); and (7) glucose transporter 1 (ab128033, Abcam; Figure S1).

Techniques: Quantitative Proteomics, Western Blot, Control

Journal: Frontiers in Marine Science

Article Title: Hypothermal Effects on Energy Supply for Ionocytes in Gills of Freshwater- and Seawater-Acclimated Milkfish, Chanos chanos

doi: 10.3389/fmars.2022.880103

Figure Lengend Snippet: FIGURE 5 | Activity levels of citrate synthase (CS) (A) and cytochrome c oxidase IV (COX IV) (B) in the gills of milkfish acclimated to 28°C (white bar) and 18°C (striped bar). Different letters in the FW milkfish (a, b) and SW milkfish (a’, b’) indicate significant differences between the normal and low-temperature groups. The x and y indicate significant differences between the FW and SW groups at 28°C or 18°C. Values represent the mean ± standard error of the mean (SEM), n = 9, P < 0.05.

Article Snippet: The following primary antibodies were used in this study: (1) Na+, K+-ATPase a1 subunit (a6F, DSHB, Iowa City, IA, USA); (2) Na+, K+-ATPase a subunit (a5, DSHB, Iowa City, IA, USA); (3) citrate synthase (sc-390693, Santa Cruz Biotechnology, Dallas, TX, USA); (4) COX IV (#4850, Cell Signaling, Beverly, MA, USA); (5) b-actin (GTX109639, Genetex, Irvine, CA, USA); (6) glycogen phosphorylase (GTX124390, Genetex); and (7) glucose transporter 1 (ab128033, Abcam; Figure S1).

Techniques: Activity Assay

Journal: Cell reports

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

doi: 10.1016/j.celrep.2018.10.058

Figure Lengend Snippet: The effect of COX7A2L absence on MRC complex assembly was investigated in two COX7A2L -KO clones, clone 1 (KO1) and clone 2 (KO2), compared with the control HEK293T cells (WT). (A) Mitochondria extracted with a digitonin/protein ratio of 4:1 (g/g) and analyzed by BN-PAGE, followed by CI- and CIV-IGA assays, or alternatively, by immunoblotting using the indicated antibodies. (B) Subsequent 2D-BN/SDS-PAGE and immunoblot analyses were performed with antibodies against COX7A2L and the indicated OXPHOS subunits. (C) To address the relative amount of CIII 2 in COX7A2L -KO cells, the signals from the CORE2 antibody from four BN-PAGE experiments were quantified by densitometry, normalized by CII, and indicated as mean ± SD. (D) BN-PAGE analyses in whole-cell extracts prepared in the presence of digitonin (detergent/protein ratio, 4:1) or 1% lauryl maltoside (LM). The CIII 2 signals were quantified and normalized by CII using the histogram function of the Adobe Photoshop program on digitalized images, and the values were expressed relative to the control. Error bars represent the mean ± SD of four independent experiments. (E) Spectrophotometric measurements of the individual activities of MRC complexes I to IV (CI–CIV) in WT and COX7A2L -KO cells. Enzyme activities are expressed as cU/U citrate synthase (CS). Error bars represent the mean ± SD of four repetitions. *p < 0.05; **p < 0.01. MegaC, megacomplexes probably containing more than one copy of CI, CIII 2 , and CIV. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ). II, complex II. Subcomplexes that contain COX1 and COX4 are indicated. Apparent subcomplexes that contain CORE2 are antibody artifacts that disappear in 2D-BN/SDS-PAGE gels. See also .

Article Snippet: COX4I1 - Myc-DDK in pCMV6-Entry , Origene , Cat# RC209374.

Techniques: Clone Assay, Control, Western Blot, SDS Page

Journal: Cell reports

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

doi: 10.1016/j.celrep.2018.10.058

Figure Lengend Snippet:

Article Snippet: COX4I1 - Myc-DDK in pCMV6-Entry , Origene , Cat# RC209374.

Techniques: Recombinant, Modification, Clear Native PAGE, Software

Journal: Metabolites

Article Title: Effect of Expression of Nuclear-Encoded Cytochrome C Oxidase Subunit 4 Isoforms on Metabolic Profiles of Glioma Cells

doi: 10.3390/metabo12080748

Figure Lengend Snippet: Metabolite profiling of U251 glioma cells overexpressing the COX4-1 or COX4-2 isoform. ( A ) Two-dimensional PCA score plots of untargeted metabolomics data from COX4-1-overexpressing glioma cells (blue) and COX4-2-overexpressing glioma cells (red). ( B ) Volcano plot of metabolomic data. The x-axis represents the mean fold change (log 2 ratio) in the relative intensity of each metabolite between the two samples (COX4-2-overexpressing relative to the COX4-1-overexpressing cells). The y-axis represents the statistical significance (-log 10 -transformed p -values) of each metabolite. Metabolites are color coded by metabolic pathway.

Article Snippet: U251 COX4-2-KO cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies, Rockville, MD, USA).

Techniques: Transformation Assay