Journal: Nature immunology

Article Title: Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16

doi: 10.1038/s41590-018-0238-4

Figure Lengend Snippet: (a–d), Binding patterns of Bcl11b, Chd4, Hdac2, Mta2, Rest, Ring1b, LSD1 and Runx1 in wildtype DN3 cells and Bcl11b -deficient CD25 + cells, with H3K27Ac ChIP-seq and RNA-seq tracks of control and Bcl11b -deficient cells. Data are representative of two independent ChIP experiments. The Id2 (a), Zbtb16 (b), Tnni1 (c) and Cd163l1 (d) loci are shown. Magenta rectangles: Bcl11b-dependent cofactor peaks. Green rectangles: cofactor peaks specifically detected in Bcl11b -deficient cells. (e, f), Differentially expressed genes in Bcl11b -deficient cells are bound by Bcl11b-dependent cofactor peaks. DEGs were from 4 independent pairs of control and Bcl11b -deleted samples; |log 2 FC|>1, FDR < 0.05, RPKM >1 . Numbers of Bcl11b-repressed, Bcl11b-dependent and non-DEGs bound by Bcl11b-dependent cofactor and H3K27Ac peaks (e), or newly generated cofactor and H3K27Ac peaks in Bcl11b -deficient cells (f) are shown. P values were determined by two-sided Fisher’s exact test. Calculations were based on ChIP-seq peaks scored as reproducible in two independent replicate samples.

Article Snippet: Six μg per 10 7 cells of anti-Bcl11b Abs (a mixture of A300–383A (Bethyl), A300–385A (Bethyl), ab18465 (Abcam) and 12120 (CST)), or anti-Chd4 Ab (A301–081A), anti-Mta2 Ab (sc-9447), anti-HDAC2 Ab (ab12169), anti-Rest Ab (12C11–1B11), anti-Ring1b Ab (A302–869A), anti-LSD1 Ab (ab17721), anti-Runx1 Ab (ab23980), or anti-H3K27Ac Ab (ab4729) were each separately pre-bound to Dynabeads anti-Rabbit, Dynabeads anti-Mouse or Dynabeads Protein A/G (Invitrogen) and then added individually to the diluted chromatin complexes in parallel aliquots.

Techniques: Binding Assay, ChIP-sequencing, RNA Sequencing, Control, Generated

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells

doi: 10.1038/s41374-018-0087-7

Figure Lengend Snippet: Genetic inactivation of Notch signaling pathway significantly diminishes BMP9-induced osteogenic differentiation of MSCs. ( a ) Restoration of PS1 expression in PS1 −/− /PS2 −/− MEFs. The PS1 −/− /PS2 −/− double-knockout (DKO) MEFs were stably transduced with retroviral vector expressing Flag-tagged PS1 (DKO-PS1) or empty vector (DKO-EV). Sunconfluent stable line cells were lysed and subjected to Western blotting analysis using antibodies against the N-terminal fragment of PS1 (PS1-NTF) (a), nicastrin (b) or β-actin (c). Each assay was done in two independent batches of experiments. Representative results are shown. b , c Double-knockout PS1 −/− /PS2 −/− significantly blunts BMP9-induced ALP activity in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP. ALP activity of the infected cells were determined either by qualitative histochemical staining at day 5 ( b ) or by quantitative chemiluminescence assay at 3, 5, or 7 days after infection ( c , panel a). ALP activity for different titers of AdBMP9 infected DKO-EV or DKO-PS1 cells, was also determined at three days after infection ( c , panel b). Each assay condition was done in triplicate. Representative results are shown. ** p < 0.001. d Double-knockout PS1 −/− /PS2 −/− significantly inhibits BMP9-induced expression of late osteogenic markers in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP for five days. Cells were fixed and subjected to immunocytochemical staining with the osteocalcin (Ocn) (a) or osteopontin (Opn) (b) antibody. Minus primary antibody staining was used as a negative control (data not shown). Each staining condition was done in duplicate. Representative results are shown. e Double-knockout PS1 −/− /PS2 −/− significantly inhibits BMP9-induced matrix mineralization in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP and maintained in the mineralization medium for 14 days. The cells were fixed and subjected to Alizarin Red S staining. Each assay condition was done in triplicate. Representative results are shown

Article Snippet: After electrophoretic separation, proteins were transferred to Immobilon-P membranes, which were blocked and incubated overnight with primary antibodies against N-terminal of PS1 (or PS1NT, homemade) [ ], nicastrin (goat, N-19, Santa Cruz Biotechnology), and β-actin (mAb, Cat# A5441, SigmaMillipore) as described [ ].

Techniques: Expressing, Double Knockout, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Western Blot, Activity Assay, Infection, Staining, Chemiluminescence Immunoassay, Negative Control

Journal: Cell reports

Article Title: Familial Alzheimer mutations stabilize synaptotoxic γ-Secretase-substrate complexes

doi: 10.1016/j.celrep.2024.113761

Figure Lengend Snippet:

Article Snippet: Rabbit anti-nicastrin , Novus Biologicals , Cat. No. NBP2-57365.

Techniques: Virus, Recombinant, Expressing, Synthesized, Mutagenesis, Enzyme-linked Immunosorbent Assay, Stable Transfection, Double Knockout, CRISPR, Plasmid Preparation, Transgenic Assay, FLAG-tag, Sequencing, Software, Fluorescence, Imaging, Microscopy, Confocal Microscopy