Journal: iScience

Article Title: SNARE protein SNAP25 regulates the chloride-transporter KCC2 in neurons

doi: 10.1016/j.isci.2024.111156

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Transferrin Receptor , Invitrogen , Cat#13–6800; RRID: AB_2533029.

Techniques: Purification, Virus, Plasmid Preparation, Recombinant, In Situ, shRNA, Software

Journal: iScience

Article Title: Regulation of the DLC3 tumor suppressor by a novel phosphoswitch

doi: 10.1016/j.isci.2024.110203

Figure Lengend Snippet: Regulation of DLC3 membrane association by a novel polybasic region (A) BH plot of basic and hydrophobic residues in DLC3 using the scale developed by Brzseska et al. The relative localization of the SAM, GAP and START domains are schematically annotated on the profile. The red box marks the identified polybasic region (PBR) spanning amino acids (aa) 199–221 with the sequence given. The blot of this region is magnified in the insert. (B) Line diagram showing the domain organization of full-length DLC3 and fragments used for the lipid overlay assay in (C), with the PBR marked in red. (C) Recombinant GST-tagged N-terminal DLC3 fragments containing the PBR (left) or lacking the PBR (right) were incubated with lipid strips. Bound protein was detected by immunoblotting with anti-GST antibody, followed by HRP-coupled secondary antibody. DAG = diacylglycerol, PA = phosphatidic acid, PS = phosphatidylserine, PE = phosphatidylethanolamine, PC = phosphatidylcholine, PG = phosphatidylglycerol, PI = phosphatidylinositol, sulfatide = 3-sulfogalactosylceramide. (D) Localization of GFP-DLC3 K725E full-length (FL) and ΔPBR in MCF7 cells inducibly expressing GFP-DLC3. E-cadherin-specific immunostainings. Images are maximum intensity projections of several confocal sections. Scale bars: 10 μm. (E and F) Analysis of images from (D). Graph shows the mean fluorescence intensity (MFI ±SEM) of the signal at cell junctions versus the cytoplasmic signal for GFP (E) or E-cadherin (F) ( n = 3; N = 50, 43 cells; t test: p = 0.0123 (E), p = 0.5194, ns = not significant (F)). (G) Biochemical fractionation of MCF7 cells stably expressing GFP-DLC3 K725E or K725E ΔPBR into soluble supernatant and membrane-containing pellet fractions. Fractions were analyzed by immunoblotting with the indicated antibodies followed by HRP-coupled secondary antibody. (H) Shown is the distribution of GFP signal in the immunoblotted fractions analyzed by Fiji, normalized to GAPDH (supernatant fraction) or transferrin receptor (pellet fraction) (line shows mean of 4 independent experiments; two-way ANOVA with Sidak’s multiple comparison test: p = 0.0147).

Article Snippet: The following antibodies were used in this study: mouse anti-α-tubulin mAb (used 1:10000 in WB, 05–829), mouse anti-FLAG M2 (1:1000 in WB, F1804) and rabbit anti-GAPDH pAb (1:15000 in WB, G9545) from Sigma-Aldrich (St.Louis, USA); mouse anti-transferrin receptor mAb (used 1:1000 in WB, 13-6800) from Invitrogen (Karlsruhe, Germany); goat anti-GST pAb (1:5000 in WB, GE27-4577-01) from GE Healthcare (Piscataway, USA); mouse anti-GFP mAb (1:250 in IF, 1:1000 in WB, 11814460001) from Roche Biosciences (Basel, Switzerland); mouse anti β-catenin mAb (1:500 in IF, 610154) from BD Biosciences (Franklin Lakes, USA); rabbit anti-E-cadherin mAb (1:200 in IF, 3195), rabbit anti-ZO-1 mAb (1:200 in IF, 13663) and rabbit anti-GFP mAb (1:1000 in WB, 2956) from Cell Signaling Technologies (Danvers, USA).

Techniques: Membrane, Sequencing, Overlay Assay, Recombinant, Incubation, Western Blot, Expressing, Fluorescence, Fractionation, Stable Transfection, Comparison

Journal: iScience

Article Title: Regulation of the DLC3 tumor suppressor by a novel phosphoswitch

doi: 10.1016/j.isci.2024.110203

Figure Lengend Snippet:

Article Snippet: The following antibodies were used in this study: mouse anti-α-tubulin mAb (used 1:10000 in WB, 05–829), mouse anti-FLAG M2 (1:1000 in WB, F1804) and rabbit anti-GAPDH pAb (1:15000 in WB, G9545) from Sigma-Aldrich (St.Louis, USA); mouse anti-transferrin receptor mAb (used 1:1000 in WB, 13-6800) from Invitrogen (Karlsruhe, Germany); goat anti-GST pAb (1:5000 in WB, GE27-4577-01) from GE Healthcare (Piscataway, USA); mouse anti-GFP mAb (1:250 in IF, 1:1000 in WB, 11814460001) from Roche Biosciences (Basel, Switzerland); mouse anti β-catenin mAb (1:500 in IF, 610154) from BD Biosciences (Franklin Lakes, USA); rabbit anti-E-cadherin mAb (1:200 in IF, 3195), rabbit anti-ZO-1 mAb (1:200 in IF, 13663) and rabbit anti-GFP mAb (1:1000 in WB, 2956) from Cell Signaling Technologies (Danvers, USA).

Techniques: Virus, Recombinant, Membrane, Mass Spectrometry, Stable Transfection, Expressing, Cloning, Mutagenesis, Real-time Polymerase Chain Reaction, Software

Journal: Biomedicines

Article Title: Role of Lipid Rafts on LRP8 Signaling Triggered by Anti-β2-GPI Antibodies in Endothelial Cells

doi: 10.3390/biomedicines11123135

Figure Lengend Snippet: Lipid microdomains localization of LRP8 and phospho-Dab2 following triggering with anti-β2-GPI antibodies. ( A ). Sucrose gradient fractions obtained from HUVECs, treated with affinity-purified anti-β2-GPI antibodies for 45 min, or left untreated, were analyzed with Western Blot using anti-LRP8 mAb, anti-phospho-Dab2 (ser24) Ab, anti-CD71 mAb or anti-flotillin Ab. Right panel. Bar graphs of densitometric analysis. The columns indicate the percentage distribution across the gel of raft fractions 4-5-6 (Triton X-100-insoluble fractions) and 9-10-11 (Tri-ton X-100-soluble fractions), as detected with scanning densitometric analysis. Results represent the mean ± SD from 3 independent experiments. **** p < 0.0001. ( B ). Fractions (4–6) (insoluble Triton X-100 fractions) and fractions 9–11 (soluble Triton X-100 fractions) were spotted onto nitrocellulose strips and analyzed via dot blot using Cholera Toxin (CTx) B Subunit-Peroxidase.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membranes (Bio-Rad), blocked with 1% BSA in TBS-T (Bio-Rad) and probed with mouse anti-LRP8 mAb (Invitrogen), rabbit anti-phospho-Dab2 (ser24) Ab (Bioss Antibodies), mouse anti-Transferrin receptor (CD71) mAb (Abcam), and goat anti-flotillin Ab (Abcam).

Techniques: Affinity Purification, Western Blot, Dot Blot