Journal: Life Science Alliance

Article Title: Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

doi: 10.26508/lsa.202101309

Figure Lengend Snippet: (A) Coomassie-stained SDS–PAGE gel of the purification steps. Lanes: M: membrane, L: load, FT: flow-through, W: wash, E: –IMAC elution, rE: reverse IMAC elution. (B) Representative size exclusion chromatography elution profile of IMAC purified MARCH5 on a Superose 6 increase 10/300 GL.

Article Snippet: Samples were subjected to SDS–PAGE (8 μL sample loaded on Mini protean TGX gels 4–20%, #4561096; Bio-Rad), and proteins were visualised by Coomassie blue staining (InstantBlue Coomassie protein stain, #ab119211; Abcam) or immunoblotting (2.5 μl sample loaded on gel) using Bio-Rad ChemiDoc Touch gel Imaging System, anti-ubiquitin (1:2,000, #NB300-130; Novusbio) and anti-MARCH5 (1:500, #ab185054; Abcam) and secondary antibody from anti-rabbit HRP (1:2,000, #7074; Cell Signalling), anti-mouse HRP (1:4,000, #NA931; GE Healthcare).

Techniques: Staining, SDS Page, Purification, Size-exclusion Chromatography

Journal: Life Science Alliance

Article Title: Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

doi: 10.26508/lsa.202101309

Figure Lengend Snippet: (A, B ) Coomassie-stained SDS–PAGE gel and (B) Western blot targeting ubiquitin (top row) and MARCH5 (bottom row) of ubiquitination assay of MARCH5 in the presence of lipids (PE, PG, PI(4)P, PC, PA, or CL) or in the absence of lipids (+ control) in a molar ratio of 1:10 protein to lipid. − control included all reaction components except MARCH5. The ubiquitination reactions were terminated at different time points (0, 15, 30, and 60 min). (C, D) Relative band intensities of MARCH5 at mono-ubiquitin at 10 kD (C) and MARCH5 at 25 kD (D) of Coomassie-stained SDS–PAGE gels. Time 0 min of each reaction solution represents 100% and reduction of either MARCH5 or mono-ubiquitin is represented relative to the respective time 0. Data information: In (A, B), experiments were performed as three independent experiments and (C, D) data are represented as mean values ± SEM of these three independent experiments. Source data are available for this figure.

Article Snippet: Samples were subjected to SDS–PAGE (8 μL sample loaded on Mini protean TGX gels 4–20%, #4561096; Bio-Rad), and proteins were visualised by Coomassie blue staining (InstantBlue Coomassie protein stain, #ab119211; Abcam) or immunoblotting (2.5 μl sample loaded on gel) using Bio-Rad ChemiDoc Touch gel Imaging System, anti-ubiquitin (1:2,000, #NB300-130; Novusbio) and anti-MARCH5 (1:500, #ab185054; Abcam) and secondary antibody from anti-rabbit HRP (1:2,000, #7074; Cell Signalling), anti-mouse HRP (1:4,000, #NA931; GE Healthcare).

Techniques: Staining, SDS Page, Western Blot, Ubiquitin Assay

Journal: Life Science Alliance

Article Title: Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

doi: 10.26508/lsa.202101309

Figure Lengend Snippet: (A, B ) Coomassie-stained SDS–PAGE gel and (B) Western blot targeting MARCH5 (left) and ubiquitin (right) of ubiquitination and de-ubiquitination assay. U: ubiquitination assay of MARCH5 in presence or absence (+control) of lipids after 120 min, D: deubiquitination of formed ubiquitin chains by MARCH5 in the presence or absence of lipids after 120 min. (C) Crosslinking experiment of MARCH5 in absence of lipids (control) and in presence of CL in a 1:10 M ratio of protein to lipid. Samples were taken after 0, 15, 30, and 60 min of incubation with the crosslinker.

Article Snippet: Samples were subjected to SDS–PAGE (8 μL sample loaded on Mini protean TGX gels 4–20%, #4561096; Bio-Rad), and proteins were visualised by Coomassie blue staining (InstantBlue Coomassie protein stain, #ab119211; Abcam) or immunoblotting (2.5 μl sample loaded on gel) using Bio-Rad ChemiDoc Touch gel Imaging System, anti-ubiquitin (1:2,000, #NB300-130; Novusbio) and anti-MARCH5 (1:500, #ab185054; Abcam) and secondary antibody from anti-rabbit HRP (1:2,000, #7074; Cell Signalling), anti-mouse HRP (1:4,000, #NA931; GE Healthcare).

Techniques: Staining, SDS Page, Western Blot, Ubiquitin Assay, Incubation

Journal: Life Science Alliance

Article Title: Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

doi: 10.26508/lsa.202101309

Figure Lengend Snippet: (A, B ) Coomassie-stained SDS–PAGE and (B) Western blot visualised by anti-ubiquitin antibodies (top row) or anti-MARCH5 antibodies (bottom row) of samples taken from the MARCH5 ubiquitination assay in presence of CL at 1:1, 1:5, 1:10, and 1:50 M ratio of protein to lipid or in the absence of lipids (+ control). − control included all reaction components except MARCH5. The ubiquitination reaction was terminated at different time points (0, 15, 30, 60, and 120 min). (C, D) Relative band intensities of MARCH5 at 25 kD (C) and mono-ubiquitin at 10 kD (D) of Coomassie-stained of SDS–PAGE gels. Time 0 min of each reaction solution represents 100%, and reduction of either MARCH5 or mono-ubiquitin is represented relative to their respective band at time 0. Data information: In (A, B), experiments were performed as three independent experiments and (C, D) data were represented as mean values ± SEM of these three independent experiments. Source data are available for this figure.

Article Snippet: Samples were subjected to SDS–PAGE (8 μL sample loaded on Mini protean TGX gels 4–20%, #4561096; Bio-Rad), and proteins were visualised by Coomassie blue staining (InstantBlue Coomassie protein stain, #ab119211; Abcam) or immunoblotting (2.5 μl sample loaded on gel) using Bio-Rad ChemiDoc Touch gel Imaging System, anti-ubiquitin (1:2,000, #NB300-130; Novusbio) and anti-MARCH5 (1:500, #ab185054; Abcam) and secondary antibody from anti-rabbit HRP (1:2,000, #7074; Cell Signalling), anti-mouse HRP (1:4,000, #NA931; GE Healthcare).

Techniques: Staining, SDS Page, Western Blot, Ubiquitin Assay

Journal: Life Science Alliance

Article Title: Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

doi: 10.26508/lsa.202101309

Figure Lengend Snippet: (A, B ) Coomassie-stained SDS–PAGE gel and (B) Western blot visualised with anti-ubiquitin antibodies (middle row) or anti-MARCH5 antibodies (bottom row) of MARCH5 ubiquitination assay in presence of PA or PC in 1:1, 1:5, 1:10, and 1:50 M ratio of protein to lipid. The ubiquitination reaction was stopped at different time points (0, 15, 30, 60, and 120 min). (C, D, E, F) Relative band intensities of MARCH5 presence of PA (C) and PC (D) and mono-ubiquitin in the presence of PA (E) and PC (F). Time 0 min of each reaction solution represents 100% and reduction of either MARCH5 or mono-ubiquitin is represented relative to the respective time 0. Data information: (A, B) Data were performed in three independent experiments and (C, D, E, F) data are shown as mean values ± SEM of these three independent experiments. Source data are available for this figure.

Article Snippet: Samples were subjected to SDS–PAGE (8 μL sample loaded on Mini protean TGX gels 4–20%, #4561096; Bio-Rad), and proteins were visualised by Coomassie blue staining (InstantBlue Coomassie protein stain, #ab119211; Abcam) or immunoblotting (2.5 μl sample loaded on gel) using Bio-Rad ChemiDoc Touch gel Imaging System, anti-ubiquitin (1:2,000, #NB300-130; Novusbio) and anti-MARCH5 (1:500, #ab185054; Abcam) and secondary antibody from anti-rabbit HRP (1:2,000, #7074; Cell Signalling), anti-mouse HRP (1:4,000, #NA931; GE Healthcare).

Techniques: Staining, SDS Page, Western Blot, Ubiquitin Assay