Journal: bioRxiv

Article Title: Rescue of pATOM36-depleted T. brucei by human MTCH1/2 reveals common features of protein insertases

doi: 10.64898/2026.02.13.705746

Figure Lengend Snippet: A) Schematic representation of the pATOM36-GFP construct used for overexpression and the TEV-digested pATOM36 obtained after the purification procedure. B) Immunofluorescence microscopy of pATOM36-GFP (green) in yeast, co-stained with MitoTracker (yellow) to visualize mitochondria. C) SDS-PAGE and immunoblot of non-digested pATOM36-GFP and TEV-digested pATOM36 following detergent solubilization and Ni-NTA purification. D) Schematic of the in vitro lipid-scrambling assay. pATOM36 is light blue, lipids gray, fluorescent NBD-PE yellow, and Triton X-100 red. E) Time-course fluorescence measurements of lipid scrambling in pATOM36 proteoliposomes (blue) versus empty liposomes (red) and CybB561 proteoliposomes (orange). Liposomal membrane integration of pATOM36 was further assessed by alkaline carbonate extraction, where reconstituted proteins sediment in the pellet (Pel) and non-reconstituted proteins remain in the supernatant (Sup), compared to total input (Inp) by SDS-PAGE and immunoblotting.

Article Snippet: Prior to transfer, gels were equilibrated 5 min in SDS-PAGE running buffer (25 mM Tris, 1 mM EDTA, 190 mM glycine, 0.05% (w/v) SDS) to facilitate protein transfer to PVDF membranes (Thermo Fisher Scientific) in 20 mM Tris, 150 mM glycine, 0.02% SDS, 20% methanol , .

Techniques: Construct, Over Expression, Purification, Immunofluorescence, Microscopy, Staining, SDS Page, Western Blot, In Vitro, Fluorescence, Liposomes, Membrane, Extraction

Journal: bioRxiv

Article Title: APP metabolism is regulated by p97/VCP through an autophagy and endolysosome-dependent mechanism

doi: 10.64898/2026.02.03.703492

Figure Lengend Snippet: (A) SY5Y-APP695 cells were transfected with scrambled siRNA (siScr) or VCP siRNA (siVCP) for 72 hours. (A) SDS-PAGE resolved cell protein lysates, and transferred membranes were blotted with antibodies targeting VCP, poly-ubiquitinated proteins (Poly-Ub), SQSTM1 (p62), APP-Cter-C17 (APP), and β-actin as a protein loading control. Apparent molecular weights are indicated to the left of the Western blots in kilodaltons (kDa) (B and C). Densitometric analyses and semi-quantification of VCP, Poly-Ub proteins, immature (mi.) and mature (mat.) APP levels, and APP α-carboxy-terminal fragments (APP-αCTF). (D) The concentrations of Aβ 1-40 and Aβ 1-42 peptides were measured in the cell-conditioned media by ELISA. Histograms represent the mean ± SD on the y-axis and are expressed as a percentage of the control value, arbitrarily set at 100%. n=6 *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 Unpaired t-test.

Article Snippet: Cell homogenates were separated on 4-12% Bis-Tris SDS-PAGE (Invitrogen) or 16% Tris-Tricine gels (Bio-Rad) for APP-CTFs analysis.

Techniques: Transfection, SDS Page, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: APP metabolism is regulated by p97/VCP through an autophagy and endolysosome-dependent mechanism

doi: 10.64898/2026.02.03.703492

Figure Lengend Snippet: (A) SY5Y-APP695 cells were transfected with negative control scrambled siRNA (siVCP -) or with VCP siRNA (siVCP +) for 72h and then treated with 40µg/mL of cycloheximide (CHX) for the indicated times from 0 to 3 hrs. Western membranes were immunoblotted with the following antibodies: APP-Cter-C17 (APP im. and APP mat.), VCP, and β-actin as a loading control. (B and C) Densitometric analyses and quantification of immature and mature APP levels were expressed as a percentage of the control (Ctrl) on the y-axis, over CHX treatment time on the x-axis. The black dotted lines represent quantification for the scrambled siRNA, while the grey dotted lines represent the siVCP condition (D). VCP siRNA reduces cell surface APP levels in SY5Y-APP695 cells. SY5Y-APP695 cells were transfected with scrambled siRNA (-) or with VCP siRNA (+) for 72h. Cell-surface proteins were chemically biotinylated and isolated with immobilized avidin beads. Flow-through and avidin-bound proteins were resolved by SDS-PAGE, and APP, VCP, and Calnexin (a cytosolic protein) were detected using antibodies against APP (APP Cter-C17), VCP, and calnexin. (E) Densitometric analysis and quantification of total APP levels. The graph indicates the mean ± SD. n=4 *p<0.05, **p<0.0, Unpaired t-test.

Article Snippet: Cell homogenates were separated on 4-12% Bis-Tris SDS-PAGE (Invitrogen) or 16% Tris-Tricine gels (Bio-Rad) for APP-CTFs analysis.

Techniques: Transfection, Negative Control, Western Blot, Control, Isolation, Avidin-Biotin Assay, SDS Page

Journal: bioRxiv

Article Title: APP metabolism is regulated by p97/VCP through an autophagy and endolysosome-dependent mechanism

doi: 10.64898/2026.02.03.703492

Figure Lengend Snippet: (A) VCP colocalizes with APP in SY5Y-C99 cells, as shown by co-labeling with APP-Cter-C17 (Red) and VCP (Green) antibodies. (B) VCP was immunoprecipitated, and C99 was detected only following C99-induced expression (C99 lane) compared to the non-induced condition (Ctrl lane). (C) SY5Y-C99 cells were treated for 24 hours with 1.5 µM of the allosteric VCP ATPase inhibitor NMS-873 as indicated. (C-D) SDS-PAGE-resolved cell protein lysates and nitrocellulose membranes were immunolabeled with the following antibodies: VCP, APP-Cter-C17, and Histone H3 as a loading control. (E) ELISA analysis of conditioned medium from SY5Y-C99 cells was performed to quantify secreted Aβ 1-40 and Aβ 1-42 . Histograms show the mean ± SD. n=3; ns=non-significant, *p<0.05, **p<0.01, ***p<0.001. Data were analyzed using an unpaired t-test or Mann-Whitney test.

Article Snippet: Cell homogenates were separated on 4-12% Bis-Tris SDS-PAGE (Invitrogen) or 16% Tris-Tricine gels (Bio-Rad) for APP-CTFs analysis.

Techniques: Labeling, Immunoprecipitation, Expressing, SDS Page, Immunolabeling, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: APP metabolism is regulated by p97/VCP through an autophagy and endolysosome-dependent mechanism

doi: 10.64898/2026.02.03.703492

Figure Lengend Snippet: (A) SY5Y-APP695 cells were treated for 24 h with autophagy activator Torin1 (1µM), autophagy flux inhibitors Bafilomycin A1 (BafA1, 100nM) or Chloroquine (CQ, 30 µM). Protein cell lysates were resolved by SDS-PAGE, transferred, and nitrocellulose membranes were blotted with APP-Cter-C17 rabbit polyclonal serum for the detection of mature (Mat.) and immature (im.) APP as well as APP carboxy-terminal fragments (APP-βCTF and APP-αCTF). β-actin staining was used as a protein loading control. Apparent molecular weight in kilodaltons (kDa) using molecular weight markers is indicated to the left of the Western blot images. Mature and immature proteoforms of APP were quantified (B) as well as α- and β-CTF (C) and Aβ 1-40 and Aβ 1-42 peptides (D). (E) SY5Y-APP695 cells were treated for 24 h with NMS-873 alone or NMS-873 together with the autophagy activator Torin1 (1µM), autophagy flux inhibitors Bafilomycin A1 (BafA1, 100nM) or Chloroquine (CQ, 30 µM). Following SDS-PAGE, mature and immature proteoforms of APP and APP-CTFs were detected with APP-Cter-C17 polysera and β-actin, used as a protein loading control. Immature and mature APP proteoforms were quantified (F) as well as APP-CTFs (G) and A β1-40 and 1-42 peptides (H). Data are represented as histograms of the mean ± SD (minimum of n = 3 independent experiments produced in triplicate for each experimental condition), ns = non-significant, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Cell homogenates were separated on 4-12% Bis-Tris SDS-PAGE (Invitrogen) or 16% Tris-Tricine gels (Bio-Rad) for APP-CTFs analysis.

Techniques: SDS Page, Staining, Control, Molecular Weight, Western Blot, Produced