Journal: Cell Death & Disease

Article Title: Rb substantially compensates for the double loss of p130 and p107 in adult but not embryonic neural stem cell lineages

doi: 10.1038/s41419-025-07815-6

Figure Lengend Snippet: IHC on brain sagittal sections in WT ( A – A”’ , E ), RbKO ( B – B”’ , F ), DKO ( C – C”’ , G ), and TKO ( D – D”’ , H ) embryos showing Hes1 and Hes3 staining throughout the DC in the latter two genotypes only. A' – D”’ Higher magnification images of boxed areas shown in ( A – D ). The majority of Hes-positive cells co-express AC-3 (but not Ki67; data not shown). I Quantification of cell counts inside the DC at E14.5. A very low number of or no Hes1+ cells were detected in WT and RbKO mice. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 biological replicates.

Article Snippet: The primary antibodies used are: Hes1 (mouse, 1:20, DSHB PCRP-HES1-2E8-s) and Hes3 (mouse, 1:20, DSHB PCRP-HES3-1A10-s), vimentin (mouse, 1:20, DSHB 40E-C-S), and PH3 (rabbit, 1:1000, Millipore 06-570).

Techniques: Staining

Journal:

Article Title: Induction of Antigen-Specific Regulatory T Cells following Overexpression of a Notch Ligand by Human B Lymphocytes

doi: 10.1128/JVI.77.20.10872-10880.2003

Figure Lengend Snippet: Transduction of EBV-LCL by Ad5/F35 Jagged-1 with subsequent activation of Notch downstream signal in T cells. (A) Results of real-time PCR showing overexpression of Jagged-1 mRNA (×64) in transduced EBV-LCL at 48 h (MOI = 100 PFU/cell) compared with that in nontransduced EBV-LCL. Data are means ± SD from three experiments. (B) Results of Western blotting showing the Jagged-1 protein of approximately 180 kDa 48 h after transduction. Human bone marrow stromal cells were used as the positive control. Shown are the results of one experiment that is representative of three. (C) Results of real-time PCR showing HES-1 and Deltex mRNA expression levels in T cells stimulated by nontransduced autologous EBV-LCL (filled columns) compared with those for autologous EBV-LCL Jagged-1 (open columns). Overexpression after stimulation with EBV-LCL Jagged-1 was observed at 12 h but not at 24 or 48 h. Data are means ± SD from three experiments. (D) Results of Western blotting showing an overexpression of the HES-1 protein (28 kDa) in T cells 24 h after stimulation with EBV-LCL Jagged-1. Shown are the results of one experiment that is representative of three. T cells/LCL-J1, T cells stimulated by autologous EBV-LCL transduced with Ad5/F35 Jagged-1; T cells/NT LCL, T cells stimulated by nontransduced autologous EBV-LCL.

Article Snippet: The anti-HES-1 antibody (clone 35) was purchased from BD Pharmingen (San Diego, Calif.).

Techniques: Transduction, Activation Assay, Real-time Polymerase Chain Reaction, Over Expression, Western Blot, Positive Control, Expressing

Journal: PLoS Biology

Article Title: Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling

doi: 10.1371/journal.pbio.2000949

Figure Lengend Snippet: (A-F) Immunofluorescence analysis showed that Sel1l is strongly expressed in 14.5 dpc pancreata maintained for 1 d (14.5 dpc + 1 d) in ALI cultures compared to 14.5 dpc pancreata (A, D), and this correlated with a drop in the expression levels of NICD (B, E) and Hes1 (C, F). (G-I) S1pr2 signalling block with 15 μM JTE013 in 14.5 dpc + 1 d ALI cultures resulted in loss of Sel1l expression (G), concomitant with maintenance of high NICD (H) and Hes1 (I) expression. (J-L) Sel1l expression was restored in the absence of S1pr2 signalling by 1 μM of the specific proteasome inhibitor MG132 in 14.5 dpc + 1 d ALI cultures (J), and this was sufficient to restore attenuation of both NICD (K) and Hes1 (L) expression. Scale bars, 50μm (A-L). For raw data, please refer to the file.

Article Snippet: Primary antibodies used were rabbit anti-Ngn3 (1:100; Acris), rat anti-E-cadherin (1:400; Zymed), rabbit anti-C-peptide (1:200; Linco), rabbit anti-Amylase (1:300; Sigma), rabbit anti-Ptf1a (1:3,000; Gift from B. Breant), rat anti-Cytokeratin19 (1:250; DSHB), mouse anti-Glucagon (1:500; Sigma), rabbit anti-Pdx1 (1:5,000; Gift from C. Wright), mouse anti-Pdx1 (1:250; DSHB), rabbit anti-Sphk (1:250; Abcam), mouse anti-Nkx6-1 (1:1,000; DSHB), mouse anti-Insulin (1:1,000; Sigma), rabbit anti-PH3 (1:500; Cell Signaling), rabbit anti-Yap1 (1:200; Cell Signaling), rabbit anti-NICD (1:100; Cell Signaling), rat anti-Hes1 (1:500; MBL-Biozol), and rabbit anti-Sel1l (1:250; Abcam).

Techniques: Immunofluorescence, Expressing, Blocking Assay

Journal: Endocrinology and Metabolism

Article Title: Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription

doi: 10.3803/EnM.2015.30.3.381

Figure Lengend Snippet: Effects of Hes6 sumoylation on Hes1-induced transcriptional suppression. (A) HeLa cells were co-transfected with pHes1 -luciferase reporter, Hes1 (20 ng), and increasing amounts of Hes6 (50, 100, 200, and 300 ng). After overnight incubation, the bioluminescence was measured using a luminometer. Each value is the mean±SEM of three independent experiments. (B) Cells were co-transfected with the pHes1 -luciferase reporter, Hes1, and Hes6 (wild type [WT] or the 2KR [K27/30R] mutant). Each value is the mean±SEM of three independent experiments ( P <0.001). (C) Cells were co-transfected with Hes1 and Hes6 and the cell lysates were immunoprecipitated using anti-Hes1 antibodies. (D) Chromatin was extracted from HeLa cells transfected with Hes1 and Hes6 constructs encoding WT or the 2KR mutant. Chromatin immunoprecipitation (ChIP) assays were performed with the indicated antibodies. a P <0.001 vs. Hes1 expression; b P <0.001.

Article Snippet: Equal amounts of total protein were incubated with 2 µg anti-Hes6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Hes1 (AbFrontier, Seoul, Korea) antibodies for 1.5 hours at 4℃ and then the samples were incubated with the protein G-Sepharose bead slurry for 1 hour at 4℃.

Techniques: Transfection, Luciferase, Incubation, Mutagenesis, Immunoprecipitation, Construct, Chromatin Immunoprecipitation, Expressing