Journal: British Journal of Pharmacology

Article Title: P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

doi: 10.1111/j.1476-5381.2011.01303.x

Figure Lengend Snippet: Atomic force microscopy (AFM) imaging of P2X receptors of various subunit compositions. (A) His6-P2X2 and His10-P2X7 were isolated from detergent extracts of cells through binding of their His tags to Ni2+-agarose beads, followed by elution with imidazole. P2X4-HA was isolated through binding of its HA tag to anti-HA immunoaffinity beads, followed by elution with HA peptide. P2X subunits were either expressed alone or in the combinations His6-P2X2+P2X4-HA (with pull-down of His6-P2X2) or P2X4-HA+His10-P2X7 (with pull-down of His10-P2X7). Eluted samples were subjected to SDS-PAGE followed by immunoblotting with either a rabbit polyclonal anti-P2X2 antibody, a mouse monoclonal anti-HA (P2X4-HA) antibody or a rabbit anti-P2X7 antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). (B) Low-magnification AFM images of a sample isolated from cells expressing P2X4-HA. The arrowheads indicate single receptors; arrows indicate double receptors. A colour-height scale is shown at the right. Scale bars, 100 nm. (C) Gallery of zoomed images of double receptors present in samples isolated from cells expressing all five subunit combinations. A colour-height scale is shown at the right. Scale bar, 50 nm. HA, haemagglutinin; PAGE, polyacrylamide gel electrophoresis.

Article Snippet: Immune complexes were captured using Protein G-Sepharose (GE Healthcare, Chalfont St. Giles, UK), and immunoprecipitates were analysed by SDS-PAGE followed by immunoblotting with either a mouse monoclonal anti-GFP antibody (Invitrogen; P2X2-GFP), a mouse monoclonal anti-HA antibody (P2X4-HA) or a rabbit polyclonal anti-P2X7 antibody.

Techniques: Microscopy, Imaging, Isolation, Binding Assay, SDS Page, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. Quercetin-specific binding proteins were captured by quercetin-Sepharose beads, and eluted fractions were resolved by SDS-PAGE. Distinct proteins in gel-eluted bands were identified using MS and validated by immunoblotting analyses and surface plasmon resonance binding assays. Specific targets were further characterized using a series of approaches, including confocal microscopy, IP, RIP, RT-qPCR, and immunoblotting analysis. Q, quercetin; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted.

Article Snippet: Quercetin was from Sigma-Aldrich, and CNBr-Sepharose 4B was obtained from GE Healthcare.

Techniques: Binding Assay, SDS Page, Western Blot, SPR Assay, Confocal Microscopy, Quantitative RT-PCR

Journal: Nature Cell Biology

Article Title: OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

doi: 10.1038/s41556-022-01039-y

Figure Lengend Snippet: a , Mouse primary fibroblasts were mock-infected or infected with MCMV-M45mutRHIM virus (multiplicity of infection (m.o.i.) = 5) for 6 h. RIPK3 protein complexes were enriched and immunoprecipitated (IP) using RIPK3 antibody-conjugated agarose beads and analysed by mass spectrometry. Functional-related or biological-related proteins are grouped in boxes. b , HEK 293T cells were transfected with the indicated constructs, and cell lysates were immunoprecipitated with V5-specific antibody. Immunoprecipitates and whole cell extracts (input) were analysed by immunoblotting with the indicated antibodies. c , Cell death kinetics of Oasl1 +/+ and Oasl1 –/– primary fibroblasts infected with MCMV-WT or MCMV-M45mutRHIM (m.o.i. = 5). Necrotic cell death was measured on the basis of the uptake of Sytox Green and quantified in real-time from 4 to 12 h.p.i. ( n = 4 biological replicates). d , Left: microscopy analysis of cell death in Oasl1 +/+ and Oasl1 –/– primary fibroblasts infected with MCMV-M45mutRHIM at 16 h.p.i. Arrows indicate cells with necrotic features after infection. Scale bar, 20 μm. Right: quantification of necrotic cell death by measuring the release of LDH from Oasl1 +/+ and Oasl1 –/– primary fibroblasts infected with MCMV-M45mutRHIM (m.o.i. = 5) for 8 h. e , Quantification of necrotic cell death by measuring intracellular ATP levels ( n = 4) and LDH release ( n = 3) in supernatants of Oasl1 +/+ and Oasl1 –/– primary fibroblasts infected with HSV-1 (m.o.i. = 5). f , Immunoblot analysis of RIPK3 and MLKL phosphorylation (P-RIPK3 and P-MLKL, respectively) in Oasl1 +/+ and Oasl1 –/– primary fibroblasts infected with MCMV-M45mutRHIM (left) or HSV-1 (right) at m.o.i. = 5 for the indicated hours. Infected cells were collected and subjected to immunoblotting with the indicated antibodies. g , Viral replication of MCMV-M45mutRHIM and HSV-1 in Oasl1 +/+ ( n = 4 for MCMV-M45mutRHIM, n = 3 for HSV-1) and Oasl1 –/– ( n = 2) primary fibroblasts were determined by plaque assays by titrating culture supernatants at the indicating time points. Data are representative of two ( b , f , g ) or three ( c – e ) independent experiments. For c – e , g , data are presented as the mean ± s.e.m. Statistical analyses were performed using two-tailed unpaired t -test ( d ) or two-way analysis of variance (ANOVA) ( c – e , g ). NS, not significant.

Article Snippet: Supernatant was collected for overnight purification using glutathione sepharose 4B beads (Cytiva).

Techniques: Infection, Virus, Immunoprecipitation, Mass Spectrometry, Functional Assay, Transfection, Construct, Western Blot, Microscopy, Phospho-proteomics, Two Tailed Test

Journal: PLoS Pathogens

Article Title: SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella -containing vacuole

doi: 10.1371/journal.ppat.1007959

Figure Lengend Snippet: (A) HeLa cells were transfected with a plasmid encoding for EGFP-SopF for 18 h and then subject to confocal fluorescence microscopy or sequential detergent fractionation. Left panel shows a representative confocal microscopy image. EGFP-SopF (greyscale), DNA (blue). Scale bar is 10 μm. Right panel shows immunoblotting analysis. Cells were collected and subject to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against GFP, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (B) As for (A) except HeLa cells were transfected with a plasmid encoding for FLAG-SopF. FLAG-SopF was detected by immunostaining (left panel) or immunoblotting (right panel) with anti-FLAG antibodies. (C) SopF partially colocalizes with actin-binding proteins found at cell adhesion sites. HeLa cells were transfected with pFLAG-SopF for 18 h, then fixed and immunostained with anti-FLAG, anti-moesin, anti-lamellipodin and anti-vasodilator-stimulated phosphoprotein (VASP) antibodies. Representative confocal microscopy images show FLAG-SopF in green and moesin, lamellipodin or VASP in red. Scale bars are 10 μm. Insets show enlargements of boxed areas.

Article Snippet: Membranes were blocked at room temperature for 1 h with Tris-buffered saline (TBS) containing 5% (w/v) skim milk powder and 0.1% (v/v) Tween-20 (TBST-milk), then incubated with the following primary antibodies overnight at 4˚C: mouse anti-FLAG M2 affinity isolated (1:2,000 dilution; Sigma), mouse anti-HA.11 ascites (1:2,000; BioLegend), rabbit polyclonal anti-GFP (1:40,000; Thermo), mouse anti-β-lactamase (clone 8A5.A10, 1:2,000 dilution; Thermo), mouse anti-Hsp27 (clone G31, 1:20,000; Cell Signaling), rabbit polyclonal anti-calnexin (1:40,000; Enzo), rabbit polyclonal anti-lamin A/C (1:5,000; Cell Signaling) or mouse anti-LAMP-1 (clone H4A3, 1:1,000 dilution; Developmental Studies Hybridoma Bank).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Fractionation, Confocal Microscopy, Western Blot, SDS Page, Immunostaining, Binding Assay

Journal: PLoS Pathogens

Article Title: SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella -containing vacuole

doi: 10.1371/journal.ppat.1007959

Figure Lengend Snippet: (A) HeLa epithelial cells were infected with S . Typhimurium wild type (WT), Δ sopF , Δ sopF pSopF (comp), Δ sopF pSopF(1–345), Δ sopF pSopF(1–367) or Δ sopF pACYC177 (empty vector) bacteria. The proportion of cytosolic bacteria was determined by CHQ resistance assay at 90 min p.i. (upper panel) or GAL8 recruitment at 1 h p.i. (lower panel, all bacteria are constitutively expressing mCherry for fluorescence detection). Upper panel: data represent the mean ± SD (n≥3 independent experiments). Lower panel: Data represent the mean ± SD (total of >600 bacteria per strain from n≥3 independent experiments). Asterisks indicate data significantly different from WT infection (one-way ANOVA with Dunnett’s post-hoc test). (B) HeLa cells were transfected with plasmids encoding for FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h. Cells were fixed and immunostained with anti-FLAG antibodies. DNA was stained with Hoechst 33342. Representative confocal microscopy images show FLAG-SopF in greyscale and DNA in blue. Scale bars are 10 μm. (C) Subcellular fractionation of transfected cells. HeLa cells were transfected with plasmids encoding for FLAG-SopF, FLAG-SopF(1–367) or FLAG-SopF(1–345) for 18 h, then collected and subjected to sequential detergent fractionation. Equal volumes of saponin-soluble, TX-100-soluble, and SDS-soluble fractions were separated by SDS-PAGE and subject to immunoblotting with antibodies against the FLAG epitope, Hsp27 (cytosol), calnexin (membranes) and lamin A/C (nucleus). Molecular mass markers are indicated on the left. Results are representative of two independent experiments. (D) C-terminal truncations of SopF lose plasma membrane association in the S . cerevisiae mss4 tet-off strain. Wild type (WT) and mss4 tet-off yeast strains were transformed with plasmids encoding for yEGFP-SopF, yEGFP-SopF C370S, yEGFP-SopF(1–367) or yEGFP-SopF(1–345) and the subcellular localization of SopF in live cells was visualized by widefield fluorescence microscopy. Representative fluorescence images are shown. Scale bars are 2 μm. The role of a potential lipidation site in SopF localization was assessed by site-directed mutagenesis of the Cys370 residue (C370S). The grey box depicts a domain of unknown function (DUF), DUF3626, spanning amino acid residues 178–338 of SopF. (E) Quantification of SopF localization in WT and mss4 tet-off yeast strains that were transformed and visualized as described in (D). Subcellular localization was categorized as cytosol, internal membrane sites (IMS), plasma membrane (PM), or IMS and PM. Results are expressed as the mean percentage of total yeast transformants (n = 300 cells from three independent transformations).

Article Snippet: Membranes were blocked at room temperature for 1 h with Tris-buffered saline (TBS) containing 5% (w/v) skim milk powder and 0.1% (v/v) Tween-20 (TBST-milk), then incubated with the following primary antibodies overnight at 4˚C: mouse anti-FLAG M2 affinity isolated (1:2,000 dilution; Sigma), mouse anti-HA.11 ascites (1:2,000; BioLegend), rabbit polyclonal anti-GFP (1:40,000; Thermo), mouse anti-β-lactamase (clone 8A5.A10, 1:2,000 dilution; Thermo), mouse anti-Hsp27 (clone G31, 1:20,000; Cell Signaling), rabbit polyclonal anti-calnexin (1:40,000; Enzo), rabbit polyclonal anti-lamin A/C (1:5,000; Cell Signaling) or mouse anti-LAMP-1 (clone H4A3, 1:1,000 dilution; Developmental Studies Hybridoma Bank).

Techniques: Infection, Plasmid Preparation, Expressing, Fluorescence, Transfection, Staining, Confocal Microscopy, Fractionation, SDS Page, Western Blot, FLAG-tag, Transformation Assay, Microscopy, Mutagenesis

Journal: Nature Communications

Article Title: Directed natural evolution generates a next-generation oncolytic virus with a high potency and safety profile

doi: 10.1038/s41467-023-39156-3

Figure Lengend Snippet: a A modified plaque formation assay was performed and cells were imaged with a fluorescence microscope 48 hours after infection (MOI = 0.5). Scale bars, 500 μm. The scale bars in the magnified images represent 100 μm. b Plaques were counted with a fluorescence microscope 48 hours after infection. Data points represent mean plaque number per well ± SD, for n = 3 biological replicates. P < 0.0001 was determined by Two-way ANOVA relative to M1-GFP. c HCT-116 cells were incubated with M1-GFP and M1-E2M at 4 °C for 1 hours. Viral RNA was quantified by qRT-PCR and presented as mean ± SD, for n = 3 biological replicates. P < 0.0001 was calculated with Two-tailed unpaired t-test. d Western blot analysis of M1-GFP and M1-E2M incubated with MXRA8-His bound to His-Tag Mouse mAb Sepharose Beads. Precipitated viral particles were detected using an anti-E1 mAb (left). Quantification of E1 expression is shown (right). Graph bars represent mean densitometry of E1 normalized to the densitometry of MXRA8 ± SD, for n = 3 biological replicates. P = 0.0060 was calculated with Two-tailed unpaired t-test. e Time course of the binding between M1 viral particles and the MXRA8 protein, as determined via BLI. f , g HeLa, HeLa-Mxra8, Hs 578 T and Hs 578T-ΔMxra8 cells were treated with M1-GFP or M1-E2M. The infection rate was determined by flow cytometry 48 hours after infection. Graph bars represent mean infection rate % ± SD, for n = 3 biological replicates. Statistical significance was calculated using Two-way ANOVA with Sidak’s multiple comparisons test relative to M1-GFP. Adjusted P values are: f vector P = 0.6199; Mxra8 P = 0.0002; g vector, P = 0.0010; ΔMxra8 P = 0.2713. n.s.: no significance, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: M1-GFP and M1-E2M (1×10 7 PFU) virus particles, 1 μg of His-Mxra8 (Sino Biological), and His-Tag Mouse mAb Sepharose Beads (Cell Signaling Technology) were incubated overnight at 4 °C in TBS containing 10 mM CaCl 2 .

Techniques: Modification, Plaque Formation Assay, Fluorescence, Microscopy, Infection, Incubation, Quantitative RT-PCR, Two Tailed Test, Western Blot, Expressing, Binding Assay, Flow Cytometry, Plasmid Preparation

Journal: iScience

Article Title: APC-driven actin nucleation powers collective cell dynamics in colorectal cancer cells

doi: 10.1016/j.isci.2023.106583

Figure Lengend Snippet:

Article Snippet: Plasmid: GFP-alone , Addgene , Cat# 54759; RRID:Addgene_54759.

Techniques: Recombinant, Modification, Saline, Stable Transfection, Expressing, Plasmid Preparation, Software, Microscopy, Chemotaxis Assay, Migration