Journal: PLoS ONE

Article Title: Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

doi: 10.1371/journal.pone.0037598

Figure Lengend Snippet: (A) Scheme of the pulldown assay. The BG-ligand is immobilized on glutathione sepharose beads via GST-SNAP. Bound receptor proteins are concentrated on the beads after incubation with the extract and washing. (B) SNAP-eDHFR WT labeled with BG-647 (1 µM) was subjected to pulldown assay in the absence or presence of 100 µM NADPH using MTX immobilized on beads (MTX-BG +) or mock beads (MTX-BG -). Bound proteins were eluted with glutathione, submitted to SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of bound proteins normalized with the input signal in each gel (Mean±SD, n = 3). # represents P = 0.03 in paired t-test.

Article Snippet: The extract was subjected to affinity chromatography using Strep-tactin sepharose column (IBA) according to the manufacturer’s instructions.

Techniques: Incubation, Labeling, SDS Page, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Epitope Mapping with Sidewinder: An XL-MS and Structural Modeling Approach

doi: 10.3390/ijms26041488

Figure Lengend Snippet: Overview of the methodological approach used for the Sidewinder pipeline, which relies on cross-linking mass spectrometry (XL-MS) data for epitope mapping. ( a ) This involves (i) the chemical cross-linking of an antibody to its cognate antigen, followed by (ii) proteolytic digestion and (iii) protein liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The resulting data, alongside sequence information for the interactors, can then be analyzed with Sidewinder (iv) to produce per-residue probability scores for the antigen of interest. This information can subsequently be used (v) for visualization and hypothesis generation. ( b ) The Sidewinder pipeline consists of Structure, Data, and Scoring modules. The Structure module takes FASTA or PDB files as input depending on existing information, predicts structures as needed, and performs molecular docking to generate an antibody–antigen model ensemble. The Data module filters input XL-MS data files based on theoretical cross-links, which are also used to search the data before annotating the model ensemble. Finally, the Scoring module utilizes the annotations to assign weights for scoring the model ensemble on a per-residue level to identify epitopes. R S = Residue Score.

Article Snippet: In order to capture the IgGs from the medium, protein G sepharose 4 Fast Flow (Cytiva, Marlborough, MA, USA) was added to the medium and incubated end-over-end at room temperature for 2 h. The beads were collected by running the medium-bead mix through a gravity flow chromatography column (Bio-Rad, Hercules, CA, USA) and washed twice with 50 mL of PBS.

Techniques: Structural Proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Sequencing, Residue

Journal: Experimental and Therapeutic Medicine

Article Title: MiR‑221 and miR‑222 regulate cell cycle progression and affect chemosensitivity in breast cancer by targeting ANXA3

doi: 10.3892/etm.2023.11826

Figure Lengend Snippet: Expression of ANXA3 is associated with chemotherapy sensitivity. (A) Transfection efficiency was assessed according to siANXA3 concentration. (B) Cell viability was evaluated in MDA-MB-231 cells treated with si-ANXA3 and adriamycin at different doses. (C) Percentage of viable MDA-MB-231 cells. Cells were treated with adriamycin (25 nM), miR-221/222 and siANXA3. (D) Cell cycle profile was analyzed using flow cytometry. Fluorescence-activated cell sorting analysis of cells transfected with control or siANXA3. (E) FACS analysis of cells transfected with adriamycin (50 nM) alone or a combination of adriamycin and siANXA3. (F) Expression of two cell cycle regulatory factors, cyclin A and cyclin B1, were evaluated using western blotting after treatment with adriamycin alone and a combination of adriamycin and siANXA3. (G) Flow cytometry and annexin V-FITC/PI labeling were used to examine apoptosis after treatment with adriamycin, siANXA3 and their combination. (H) Expression of apoptotic factors was evaluated by western blotting after treatment with adriamycin alone and a combination of siANXA3. (I) Overall survival was evaluated according to ANXA3 levels using the Kaplan-Meier Plotter software. * P<0.05, ** P<0.01 and *** P<0.001. ANXA3, annexin A3; FACS, fluorescence-activated cell sorting; Adr, adriamycin; SRC, scrambled negative control; siANXA3, small-interfering RNA targeting ANXA3; siCTL, small-interfering RNA negative control; Asy, asynchronous; PARP, poly (ADP-ribose) polymerase; anti-miR-CTL, anti-miR 221/222 negative control; miR, microRNA.

Article Snippet: The primary antibodies were: Anti-annexin A3 (1:1,000; cat. no. ab33068; Abcam); anti-cyclin D1 (1:500; cat. no. sc-20044; Santa Cruz Biotechnology, Inc.); anti-CDK4 (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-56277); anti-cyclin B1 (1:500; cat. no. sc-245; Santa Cruz Biotechnology, Inc.); anti-cyclin A (1:500; cat. no. sc-274682; Santa Cruz Biotechnology, Inc.); anti-α tubulin (1:1,000; cat. no. sc-5286; Santa Cruz Biotechnology, Inc.); anti-β-Actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.); apoptosis western blot cocktail (1:250; cat. no. ab136812; Abcam); and total poly ADP ribose polymerase (1:1,000; cat. no. 9542; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Concentration Assay, Flow Cytometry, Fluorescence, FACS, Western Blot, Labeling, Software, Negative Control, Small Interfering RNA