Journal: Biochemistry and Biophysics Reports

Article Title: Identification of a novel MIPEP splice variant with altered substrate-binding properties

doi: 10.1016/j.bbrep.2025.102329

Figure Lengend Snippet: ΔMipep is a splice variant of Mipep . (A) Sequences from exon 14 to 17 and the exon structures of mouse Mipep and ΔMipep . The shaded area represents exons 15 and 16. (B) The domain structure of MIPEP and ΔMIPEP. Amino acids 493, 497, and 522 are zinc-binding sites. (C) The positions of primers for semiquantitative RT-PCR analysis of Mipep and ΔMipep cDNAs. (D) Agarose gel electrophoresis images of semiquantitative RT-PCR for full-length Mipep and ΔMipep in mouse tissues. Full-length Mipep plasmid (pMXs-AMNN- Mipep -Puro) and ΔMipep plasmid (pMXs-AMNN- ΔMipep -puro) were used as positive controls of full-length Mipep and ΔMipep , respectively. (E) The positions of primers and the probe for quantitative RT-PCR analysis of Mipep and ΔMipep cDNAs. (F) Copy numbers of full-length Mipep and ΔMipep mRNAs per total RNA μg in mouse tissues. Values are shown as the mean ± standard deviation (n = 3 or 4).

Article Snippet: Membranes were blocked for 60 min at room temperature using 2.5 % skim milk and 0.25 % BSA in TBS-T, and incubated overnight at 4 °C with the primary antibodies Primary antibodies against mouse MIPEP (produced as stated below), SIRT3 (5490, Cell Signaling Technology (CST), Beverly, MA, USA), MDH2 (8610, CST), COX4 (4844, CST), and LaminB1 (PM064, MBL, Tokyo, Japan) were used.

Techniques: Variant Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

Journal: Biochemistry and Biophysics Reports

Article Title: Identification of a novel MIPEP splice variant with altered substrate-binding properties

doi: 10.1016/j.bbrep.2025.102329

Figure Lengend Snippet: Changes in molecular sizes of MIPEP substrates in Mipep knockout cells overexpressing full-length Mipep or ΔMipep. (A) The lack of MIPEP was confirmed in Mipep knockout (KO) cells. (B) Full-length MIPEP and ΔMIPEP protein levels in mock, full-length Mipep - and ΔMipep -overexpressing 3T3-L1 and Mipep KO cells. ∗ indicates a non-specific band. (C) Sirtuin 3 (SIRT3), malate dehydrogenase 2 (MDH2) and cytochrome c oxidase 4 (COX4) protein levels in mock, full-length Mipep - and ΔMipep -overexpressing cells. ∗ indicates the higher molecular weight bands, while ∗∗ indicates the lower molecular weight bands. LaminB1 was used as a loading control.

Article Snippet: Membranes were blocked for 60 min at room temperature using 2.5 % skim milk and 0.25 % BSA in TBS-T, and incubated overnight at 4 °C with the primary antibodies Primary antibodies against mouse MIPEP (produced as stated below), SIRT3 (5490, Cell Signaling Technology (CST), Beverly, MA, USA), MDH2 (8610, CST), COX4 (4844, CST), and LaminB1 (PM064, MBL, Tokyo, Japan) were used.

Techniques: Knock-Out, Molecular Weight, Control

Journal: STAR Protocols

Article Title: Protocol for Isolation of Golgi Vesicles from Human and Animal Hearts by Flotation through a Discontinuous Sucrose Gradient

doi: 10.1016/j.xpro.2020.100100

Figure Lengend Snippet: Analysis of Isolated Fractions (A) Western blot of Golgi (GM130 and MYLK4), nucleus (nucleolin and MYLK4), cytoplasmic (MYLK4), and mitochondrial (NDUFB9) markers in the three fractions collected after the sucrose gradient ultracentrifuge. F1, fraction 1 (top); F2, fraction 2 (half); F3, fraction 3 (bottom). (B) Visualization of isolated Golgi vesicles using transmission electron microscopy (white arrows). Numerous smooth vesicles are seen, mostly formed by a single membrane, apparently empty with little dense content, mainly a light network of granular material. The presence of mitochondrial remains is also observed (black arrow). Bar represents 500 nm.

Article Snippet: Nucleolin primary mouse monoclonal antibody , Merk Milipore , 05-565.

Techniques: Isolation, Western Blot, Transmission Assay, Electron Microscopy, Membrane