Journal: International journal of cancer

Article Title: Testicular germ cell tumour cells release microRNA-containing extracellular vesicles that induce phenotypic and genotypic changes in cells of the tumour microenvironment.

doi: 10.1002/ijc.34697

Figure Lengend Snippet: FIGURE 1 TGCT cells secrete extracellular vesicles (EVs) into culture media. (A) Visualisation of representative normal and TGCT-derived EVs by transmission electron microscopy (TEM). (B) EV proportion (%) of a specific size, as assessed by Nanoparticle Tracking Analysis (NTA), in different cell lines. (C) EV size comparison of normal and TGCT-derived EVs based on mode. N = 3 for each condition. Measurement of EV size by NTA. (D) Protein expression of EV and cellular markers in control and TGCT cells and their derived EVs. Detection of EV (PDCD6IP and CD63), cellular [Cytochrome C1 (CYC1) and THOC1], and protein loading control (ACTB) markers by western blot. Cell lines used were Hs1.Tes (Normal/Control), TCam2 (SEM), GCT44 and 1411H (YST), and 2102Ep and NCCIT (EC). EC, embryonal carcinoma; SEM, seminoma; YST, yolk sac tumour.

Article Snippet: Immunoblots were probed with primary antibodies against programmed cell death 6 interacting protein (PDCD6IP, commonly termed ‘Alix’; Santa Cruz, catalogue: sc53540; concentration: 1:200), CD63 (Invitrogen, 10628D; 1:200), Cytochrome C1 (CYC1; Santa Cruz, sc-514 435; 1:100), THO complex 1 (THOC1, commonly termed ‘p84’; Abcam, ab487; 1:1000) and beta actin (ACTB; Abcam, ab6276; 1:5000).

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Comparison, Expressing, Control, Western Blot

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