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: The FASEB Journal

Article Title: Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes

doi: 10.1096/fj.201700014R

Figure Lengend Snippet: XPA chromatin accumulation in HGPS cells is age-dependent and regulated by ATR. A) Chromatin association assay of XPA binding in BJ and young (P13) and old (P18) HGPS fibroblasts. PARP was used as a control for the nuclear fraction, and histone 3 was used as a control for the chromatin-bound protein. B) The amount of chromatin-bound XPA was quantified in HGPS cells of 4 different ages (top) and normalized to the free XPA in the cell. C) ATR but not ATM kinase dependence of XPA chromatin association in old-age HGPS fibroblasts. The cells were treated with 10 μM ATR or ATM inhibitor for 1 h before chromatin isolation. Alternatively, the ATR/ATM inhibitor treatment was followed by 20 J/m2 UV-C treatment, followed by a 2-h recovery, to demonstrate the effectiveness of the inhibitors. Whole-cell lysates were generated and analyzed.

Article Snippet: Proteins were transferred to PVDF membranes (Santa Cruz Biotechnology) and immunoblot analysis was performed with primary antibodies directed against XPA (MA5-13835; Thermo Fisher Scientific), lamin A/C (2032; Cell Signaling Technology), GAPDH (MA5-15738; Thermo Fisher Scientific), β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA), PCNA (sc-9857; Santa Cruz Biotechnology; and ab29; Abcam), MCM7 (sc-9966; Santa Cruz Biotechnology), Rad50 (sc-18291; Santa Cruz Biotechnology), poly ADP ribose polymerase (PARP)-1 (9542; Cell Signaling), p53 (sc-47698; Santa Cruz Biotechnology), phospho-p53 (Ser15) (9286; Cell Signaling), ribonucleotide reductase catalytic subunit (RRM)-1 (sc-11733; Santa Cruz Biotechnology), RRM2 (sc-10846; Santa Cruz Biotechnology), p53RRM2 (sc-10840; Santa Cruz Biotechnology), and polymerase δ (Polδ, sc-17776; Santa Cruz Biotechnology), followed by HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Histone Association Assay, Binding Assay, Isolation, Generated

Journal: The FASEB Journal

Article Title: Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes

doi: 10.1096/fj.201700014R

Figure Lengend Snippet: XPA stalls induction of apoptosis in old progeroid cells. A) HGPS cells at P8 (young) and P20 (old) passages were transfected with scrambled sequence (siCtrl) or an XPA-specific siRNA (siXPA). Five days after transfection, the cells were harvested. Cell survival was measured by flow cytometry using TMRE, which is an indicator of cells with functional, polarized mitochondria (left). Western blot analysis demonstrated caspase-3 cleavage and confirmed XPA-knockdown efficiency (right). B) HeLa, XPAWT and XPA−/− cells from a patient with xeroderma pigmentosum were transfected with plasmid expressing SSIM (a mutant lamin A that cannot be farnesylated), progerin (LAΔ50, GFP-LAΔ50), or an empty parent vector (GFP). At 24 h after transfection, the cells were harvested for analysis by Western blot analysis. The top blot was probed with an antibody against lamin A/C. The bottom blot was probed with an antibody against PARP. C) XPAWT and XPA−/− cells were transfected with the plasmid expressing progerin (LAΔ50) or empty parent vector (GFP). Cells were harvested 24 h after transfection. These cells also were separately treated with CPT for 6 h before assay. The caspase-3 activity was measured using a fluorogenic substrate (Ac-DEVD-AFC). The relative fluorescence units were normalized to the protein concentration of the sample.

Article Snippet: Proteins were transferred to PVDF membranes (Santa Cruz Biotechnology) and immunoblot analysis was performed with primary antibodies directed against XPA (MA5-13835; Thermo Fisher Scientific), lamin A/C (2032; Cell Signaling Technology), GAPDH (MA5-15738; Thermo Fisher Scientific), β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA), PCNA (sc-9857; Santa Cruz Biotechnology; and ab29; Abcam), MCM7 (sc-9966; Santa Cruz Biotechnology), Rad50 (sc-18291; Santa Cruz Biotechnology), poly ADP ribose polymerase (PARP)-1 (9542; Cell Signaling), p53 (sc-47698; Santa Cruz Biotechnology), phospho-p53 (Ser15) (9286; Cell Signaling), ribonucleotide reductase catalytic subunit (RRM)-1 (sc-11733; Santa Cruz Biotechnology), RRM2 (sc-10846; Santa Cruz Biotechnology), p53RRM2 (sc-10840; Santa Cruz Biotechnology), and polymerase δ (Polδ, sc-17776; Santa Cruz Biotechnology), followed by HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Transfection, Sequencing, Flow Cytometry, Functional Assay, Western Blot, Plasmid Preparation, Expressing, Mutagenesis, Activity Assay, Fluorescence, Protein Concentration

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