tris hcl sds page gels  (Bio-Rad)

Journal: Toxins

Article Title: Cholera Toxin-Mediated Targeting of Botulinum Neurotoxin Activity to Pain-Associated Sensory Neurons

doi: 10.3390/toxins18040174

Figure Lengend Snippet: Production of ChoBot: ( a ) Schematic showing self-assembly of the AB5-linker, a CTB pentamer assembled around CTA2 fused to Linker 1, CTA2-cholera toxin A2 subunit, CTB-cholera toxin B subunits; ( b ) coomassie-stained SDS-PAGE gel showing that mixing the linker 1-AB5 and the light chain/translocation domain (LcTd) of BoNT/A attached to Linker 2 (LcTd-Linker 2) in the presence of Linker 3 (not visible due to low MW) results in formation of LcTd-Linkers-CTA2 (arrowhead). CTB5 is denoted as a pentamer of approx. 50 kDa. Note, the CTB5 and CTA2 association is not SDS-resistant and therefore only the linking of LcTd to CTA2 is visible as a slight shift in MW of LcTd-Linker 2. Boiling for 2 min in the SDS-PAGE sample buffer results in dissociation of CTB5 pentamer into monomeric B subunits. Molecular weight standards are denoted as MW; ( c ) schematic of ChoBot modelled using known X-ray structures of the BoNT/A, the SDS-resistant SNARE linking complex and cholera toxin (PDBs 3BTA, 1SFC and 1XTC, respectively).

Article Snippet: Cell culture medium was then removed and cells were lysed in 45 μL of SDS-PAGE running buffer (56 mM sodium dodecyl sulphate, 0.05 M Tris-HCl, pH 6.8, 1.6 mM EDTA, 6.25% glycerol, 0.0001% bromophenol blue, 10 mM MgCl2, 26 U/mL benzonase) by shaking at 900 rpm for 10 min. Lysed cells were then boiled at 95 °C for 3 min and then run on 12% Novex SDS–PAGE gels (Invitrogen) for 3 h at 4 °C to increase separation between cleaved and intact SNAP-25.

Techniques: Staining, SDS Page, Translocation Assay, Molecular Weight

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