Journal: International Journal of Oncology

Article Title: Identification of prognostic biomarkers for glioblastomas using protein expression profiling

doi: 10.3892/ijo.2011.1302

Figure Lengend Snippet: The 78 antibodies used for immunohistochemical study.

Article Snippet: X47 , VEGFR1 , Novus Biologicals, NB100–685 , 1:10 , Microwave , C, M , 62 , C, M, N.

Techniques: Immunohistochemical staining, Expressing

Journal: Current protocols

Article Title: Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro.

doi: 10.1002/cpz1.70107

Figure Lengend Snippet: Figure 15 THP-1 dramatic polarization toward an M1-like phenotype with Sepiapterin treatment in the 3D Flipwell coculture system.(A) Confocal images of THP-1 cells cocultured with Caco-2:HT- 29 in the 3D Flipwells and stained for pan macrophage marker CD68 (red) and CD80 (green). Pan macrophage CD68 marker is present in both the SEP treated sample and the untreated control. CD80 marker, typical of the M1 polarization phenotype, is more prominently present in the SEP treated sample with long pseudopodia extending outward typical of the M1 phenotype. (B) Con- focal images of THP-1 cells cocultured with Caco-2:HT-29 in the 3D Flipwell and stained for pan macrophage marker CD68 and CD163 to validate phenotypic polarization toward M1 phenotype and away from M2 phenotype. Scale bar = 25 μm.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Anti-human MUC2 (996/1) (Thermo Fisher, cat. no. MA5-12345) Anti-human CK20 (Life Technologies, cat. no. PA5-82875) Anti-human CD80 (Cell Signaling Technologies, E3Q9V, cat. no. 15416) Anti-human CD163 (Abcam, cat. no. 156769) or anti-human CD68 (Novus Biologicals, cat. no. NB100-683) Secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, cat. no. A11008) for CD80 and CK20 Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher, cat. no. A11005) for MUC2 and CD68 or CD163 DAPI, VectaShield mounting medium (Vector Laboratories, cat. no. H-1000) 50-ml conical tubes (SpectraTube, Alkali Scientific cat. no. C2750) Plastic wrap Lab tape or shipping tape Tweezers 12-well tissue culture plate (Corning Costar, cat. no. 3513) 6-well dish Aluminum foil Scalpel Round cover slips, #1, 1 oz (Corning Float Glass, cat. no. 50143822, or Fisherbrand cat. no. 12-546-2 25CIR-2) Laboratory marker (VWR, cat. no 95042-566) Attofluor cell chamber coverslip holder (Thermo Fisher Scientific, cat. no. A7816) Confocal microscope and software [e.g., Leica Microsystems TCS SP5, multi-photon laser scanning, and Suite Advanced Fluorescence (LAS AF) software] Deep Petri dish bottoms, 100 × 25–mm deep polystyrene stackable Petri dish, sterile (USA Scientific, cat. no. 8609-0625) 10-, 200-, and 1000-μl pipet tips (Alkali Scientific PurePoint or equivalent) Beckman Coulter Allegra 6 centrifuge with a swing bucket rotor for 50-ml conical tubes Optional: ImmunoPen (Fisher Scientific, cat. no. 4021761EA) Wash, permeabilization, disassembly, and blocking steps 1.

Techniques: Staining, Marker, Control

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 2. Trim32 is recruited to spindle poles during mitosis through Cdk1/cyclin B phosphorylation signaling. A,representative images of localization of Trim32 to spindle poles during mitosis but not in interphase in SH-SY5Y, TGW, and SK-N-DZ cells. Centrosome marker pericentrin, green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. B and C, representative images of Trim32 localization to spindle poles during mitosis in TGW and SK-N-DZ cells upon exposure to 5 mmol/L RO-3306 for 3 hours. DMSO (0.1%)-treated cells were used as a control. Centrosome (centriole) marker centrin-2, is green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. D, immunoblot of cyclin B expression in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Immunoblot of b-actin served as a loading control. E, representative images of Trim32 localization to spindle poles during mitosis in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Centrin-2, green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. F, percentage of cells with Trim32 localization to spindle poles in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Error bars, SEM from three experiments. Localization statuses of Trim32 at spindle poles were categorized into three types (intense, weak, or no localization). G, representative images of Flag-Trim32 or Flag-Trim32/3A localization to spindle poles during mitosis in SK-N-DZ cells transfected with Flag-Trim32 or Flag-trim32/3A expression vector. Pericentrin, green; Flag, red; DAPI (DNA), blue. Arrows, spindle poles. Flag-Trim32 localizes to spindle poles, but Flag-Trim32/3A does not. Scale bars, 10 mm.

Article Snippet: The primary antibodies used were as follows: antiNuMA antibody (NB500-174; Novus Biologicals), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (#9405S; Cell Signaling Technology), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Fbxw7 antibody (ab71961; Abcam), anti-Huwe1 antibody (ab70161; Abcam), anti-pericentrin antibody (NB100-68277; Novus Biologicals), anti-centrin 2 antibody (sc-2793R; Santa Cruz Biotechnology), anti-phospho-T58 Myc antibody (ab28842; Abcam), anti-phospho-S62 Myc antibody (ab51156; Abcam), anti-c-Myc antibody (9E10, MA1-980; Thermo Scientific), and anti-Flag antibody (#2368S; Cell Signaling Technology).

Techniques: Phospho-proteomics, Marker, Control, Western Blot, Expressing, Transfection, Plasmid Preparation