Journal: eLife

Article Title: BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

doi: 10.7554/eLife.18108

Figure Lengend Snippet: ( A ) Immunoblotting analysis of NICD1 and Notch1 (N TM ) in cortical extracts derived from WT and Bloc1s2 −/− mice at E14.5 . The results showed that the levels of NICD1 and Notch1 (N TM ) were significantly increased in the Bloc1s2 −/− cortex lysates. ( B ) Quantification of NICD1 and Notch1 (N TM ) relative to WT cortex. Data are presented as fold change normalized to the mean of WT ± s.e.m. ***p<0.001 (Student’s t-test); n=9 mice. ( C ) Immunohistochemistry staining of Notch1 on sections of WT and Bloc1s2 −/− brains at E14.5. Insets are high-magnification images of the boxed regions. Increased Notch1 expression was observed in Bloc1s2 −/− cortices at E14.5. Scale bar: 50 μm. ( D ) Quantification of signal intensity of Notch1. **p<0.01; n=9 mice. ( E - G ) qRT-PCR analysis for Hes1, Hes5 and Fabp7 of the WT and Bloc1s2 −/− dissected cortices at E14.5. The expression of Hes1, Hes5 and Fabp7 were significantly higher in the Bloc1s2 −/− cortex. Data are presented as fold change normalized to the mean of WT. *p<0.05; ***p<0.001; n=9 mice. ( H ) Immunoblot analysis of Fabp7, Hes1 and Hes5 in cortical extracts derived from WT and Bloc1s2 −/− mice at E14.5 ). ( I ) Quantification of Fabp7, Hes1 and Hes5 relative to WT cortex. Data are presented as fold change normalized to the mean of WT. ***p<0.001; n=9 mice. DOI: http://dx.doi.org/10.7554/eLife.18108.006 10.7554/eLife.18108.007 Figure 3—source data 1. Western blots of . DOI: http://dx.doi.org/10.7554/eLife.18108.007 10.7554/eLife.18108.008 Figure 3—source data 2. Western blots of . DOI: http://dx.doi.org/10.7554/eLife.18108.008

Article Snippet: Other antibodies used in this study were as follows: rabbit anti-Notch1 (1:2000, Cell Signaling Technology, 3608), rabbit anti-Fabp7 (1:5000, Millipore, ABN14), rat anti-Hes1 (1:1000, MBL, D134-3), rabbit anti-Hes5 (1:1000, Millipore, AB5708), mouse anti-EEA1 (1:1000, BD Biosciences, 610457), mouse anti-CD63 (1:1000, Millipore, NG1944051), mouse anti-GM130 (1:1000, BD Biosciences, 610822), mouse anti-Flag (1:5000, Sigma, F3165), rabbit anti-Myc (1:5000, Sigma, C3956), mouse anti-β-actin (1:10000, Sigma, A5441), anti-Runx1 (1:200, AnaSpec, 55593), anti-Notch1 (1:200, Abcam, ab65297), anti-EGFR (1:5000, Fitzgerald, 20R-ER004), anti-Cathepsin D (1:1000, SantaCruz, sc-6486).

Techniques: Western Blot, Derivative Assay, Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

doi: 10.7554/eLife.18108

Figure Lengend Snippet: ( A ) qRT-PCR analysis for Hes1 of WT and Bloc1s2 −/− MEFs incubated with DAPT or DMSO (CTRL). Data are presented as fold change normalized to the mean of WT. ***p<0.001 (Student’s t-test; n=9). ( B ) Representative images of neurospheres derived from E14.5 NPCs (WT-Ctrl, KO-Ctrl and KO-DAPT) are shown. Quantification of neurosphere diameters (μm): 58.49 ± 2.34 for WT-Ctrl, 110.15 ± 3.84 for KO-Ctrl, 60.56 ± 2.53 for KO-DAPT. ***p<0.001 (n=3 mice). ( C ) Immunostaining of neurospheres derived from WT-Ctrl, KO-Ctrl and KO-DAPT NPCs with antibodies against Nestin (NPC marker) or Tuj1 (neuron marker). ( D and E ) Quantification of the percentage (%) of Nestin-positive (proliferation) and Tuj1-positive (differentiation) cells in cultured neurospheres. 41.28 ± 1.31 (Nestin + ), 33.66 ± 2.85 (Tuj1 + ) for WT-CtrlL; 81.56 ± 2.49 (Nestin + ), 11.68 ± 1.06 (Tuj1 + ) for KO-Ctrl; 36.98 ± 1.15 (Nestin + ), 27.09 ± 1.13 (Tuj1 + ) for KO-DAPT. ***p<0.001 (Student’s t-test; n=3 mice). All graphs are mean ± s.e.m. Scale bars: 100 μm in (B); 50 μm in (C). DOI: http://dx.doi.org/10.7554/eLife.18108.014

Article Snippet: Other antibodies used in this study were as follows: rabbit anti-Notch1 (1:2000, Cell Signaling Technology, 3608), rabbit anti-Fabp7 (1:5000, Millipore, ABN14), rat anti-Hes1 (1:1000, MBL, D134-3), rabbit anti-Hes5 (1:1000, Millipore, AB5708), mouse anti-EEA1 (1:1000, BD Biosciences, 610457), mouse anti-CD63 (1:1000, Millipore, NG1944051), mouse anti-GM130 (1:1000, BD Biosciences, 610822), mouse anti-Flag (1:5000, Sigma, F3165), rabbit anti-Myc (1:5000, Sigma, C3956), mouse anti-β-actin (1:10000, Sigma, A5441), anti-Runx1 (1:200, AnaSpec, 55593), anti-Notch1 (1:200, Abcam, ab65297), anti-EGFR (1:5000, Fitzgerald, 20R-ER004), anti-Cathepsin D (1:1000, SantaCruz, sc-6486).

Techniques: Quantitative RT-PCR, Incubation, Derivative Assay, Immunostaining, Marker, Cell Culture