Journal: Mediators of Inflammation

Article Title: The Role of EGFR/PI3K/Akt/cyclinD1 Signaling Pathway in Acquired Middle Ear Cholesteatoma

doi: 10.1155/2013/651207

Figure Lengend Snippet: The cultured cells grew into monolayer and showed a polygonal “cobblestone-like” appearance (a). The cultured cells showed a positive reaction to cytokeratins by immunofluorescence, and the signal was detected in cytoplasm (b).

Article Snippet: The cells were then incubated with primary mouse antibody against cytokeratins (1 : 150 dilution; Boster, China) at 37°C for 90 min. After incubation, the cells were washed in PBS and incubated with TRITC-labeled goat-anti-mouse secondary antibody (1 : 50 dilution; Boster, China) at 37°C for 60 min. Coverslips were finally mounted for observation.

Techniques: Cell Culture, Immunofluorescence

Journal: Neurogenesis

Article Title: Characterization of Np95 expression in mouse brain from embryo to adult: A novel marker for proliferating neural stem/precursor cells

doi: 10.4161/23262133.2014.976026

Figure Lengend Snippet: Np95 is expressed abundantly in the brain at midgestation, when a large number of NS/PCs exist. ( A ) Confirmation of antibody reactivity to mouse Np95. HEK293T cells were transfected with FLAG-tagged Np95 expression plasmid and immunoblotted with anti-FLAG, -Np95 and -UHRF1 antibodies. ( B ) Western blot analysis of mouse whole brain at various stages of development, from E11 to postnatal day 7 (P7) and in 8-week-old adults, using anti-DNMT1, -Np95 and -actin antibodies. Np95 and DNMT1 bands were strongest in E11 brains. ( C ) Representative immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bar: 250 μm. ( D ) Confocal immunofluorescence images for Np95, Ki67 and Sox2 in E14 mouse forebrain sections. Scale bars: 10 μm. ( E–G ) Representative immunofluorescence images of E14 mouse forebrain sections. Immunostaining of Np95 (red), Nestin (cyan) and Ki67 ( E ), DCX (F) or MAP2ab ( G ) (green). The insets in Merge images show Hoechst staining. Np95 expression was observed only in Ki67+ or Nestin+ proliferating NS/PCs. Scale bars: 100 μm. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate.

Article Snippet: The protein samples were separated in gradient (5–20%) polyacrylamide gels (e-PAGEL; ATTO, 2331830), transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, RPN303F), and probed with anti-FLAG (1:2000; Sigma, F1804), anti-Np95 (1:2000), anti-UHRF1 (1:2000; Santa Cruz Biotechnology, sc-98817), anti-DNMT1 (1:2000; Cosmo Bio, BAM-70-201-EX) or anti-actin (1:2000; Abcam, ab3280) antibody.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Immunofluorescence, Immunostaining, Staining

Journal: International Journal of Oncology

Article Title: TIP60 governs the auto-ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: TIP60 interferes with UHRF1-USP7 association and their expression levels. HeLa cells were transfected with either TIP60-eGFP WT or TIP60ΔMYST-eGFP mutant. Western blot and immunoprecipitated samples were resolved by SDS-PAGE and immunoblotted with anti-UHRF1, anti-USP7 and anti-DNMT1 antibodies. (A) Effect of TIP60 on UHRF1, USP7 and DNMT1 levels with or without MG-132 treatment, respectively. (B and C) Quantification of the effect of TIP60 on UHRF1, USP7 and DNMT1 levels with or without MG-132 treatment, respectively. Values are the mean ± SEM from at least three independent experiments which were analyzed statistically by one-way ANOVA with Tukey's post hoc test ( * P<0.05; *** P<0.001 vs. control group). (D) Anti-UHRF1 antibody was used to co-immunoprecipitate UHRF1 and its partner USP7. (E) In a reciprocal experiment, anti-USP7 antibody was used to co-immunoprecipitate USP7 and UHRF1. Inputs and IP gels were processed in parallel under similar conditions. UHRF1, ubiquitin-like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa; USP7, ubiquitin-specific-processing protease 7; DNMT1, DNA methyltransferase 1.

Article Snippet: Other antibodies used included rabbit polyclonal anti-HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti-DNMT1 (1:5,000; cat. no. PTG-MAB0079, ProteoGenix), mouse monoclonal anti-ubiquitin (1:500; cat. no. 05-944, Sigma-Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002-1-Ig, Proteintech Group, Inc.; and cat. no. A-11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti-GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti-GFP (1:1,000; cat. no. 66002-1-Ig, Proteintech Group, Inc.), mouse monoclonal anti-p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti-caspase-3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti-BCL2 (1:1,000; cat. no. 05-826, Merck KGaA), mouse monoclonal anti-poly(ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 51-6639GR, BD Biosciences) and rabbit polyclonal anti-BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Expressing, Transfection, Mutagenesis, Western Blot, Immunoprecipitation, SDS Page