Journal: The Journal of Cell Biology

Article Title: Analysis of Notch Lacking the Carboxyl Terminus Identified in Drosophila Embryos

doi:

Figure Lengend Snippet: NΔCterm is associated with Dl during embryogenesis. (a) Autoradiographs of Western blots showing the form of N complexed with Dl in 3–6-h Canton S embryonic extracts is NΔCterm. Dl containing complexes were immunoprecipitated with mAb 202 anti-Dl antibody and the Western blots probed with the indicated N antibodies. N in the 0–3-h complexes is recognized by αNI (lane 5), αNPCR (lane 7), and αNT (lane 9), but N in the 3–6-h complexes is recognized by αNI (lane 6), αNT (lane 10), and not by αNPCR (lane 8). N was not detected on the blots when the cross-linker was not used (lanes 1 and 2) or when the anti-Dl antibody was not used (lanes 3 and 4; an anti-βGalactosidase monoclonal antibody was used instead). Thus, N complexed with Dl in 3- and 6-h embryonic extracts is NΔCterm. All N/Dl complexes shown migrate in the ∼350-kD range. Presence or absence of the carboxyl terminus did not measurably affect the electrophoretic mobility of N/Dl complexes as cross-linking renders the complexes partially resistant to denaturation during SDS-PAGE (see ). (b) Autoradiographs of Western blots showing that the level of NFull is low in 3- and 6-h and 8–11-h Canton S embryonic extracts (compare lanes 2, 5 with 1, 3, 4). The same amount of total proteins (quantitated by absorbance values at 280 nM and the BioRad D C protein assay kit) was loaded in all the lanes. Note similar levels of N350.2 and NΔCterm in lanes 1 and 2. The two blots are exposed to film for different periods. 4.25% SDS-PAGE gels were used for all Western blots. N containing complexes and N forms shown migrate in the ∼350-kD range. IP-Ab, antibody used for immunoprecipitations; W-Ab, antibody used in Western blot analysis; AEL, after egg laying; Cross-linker, Bis(sulfosuccinimidyl) suberate (Pierce).

Article Snippet: The amount of proteins in different extracts was standardized using absorbance values at 280 nM and the BioRad D C protein assay kit.

Techniques: Western Blot, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: Analysis of Notch Lacking the Carboxyl Terminus Identified in Drosophila Embryos

doi:

Figure Lengend Snippet: nd 3 embryos overexpress N molecules resembling NΔCterm, NΔCterm TMintra , and NΔCterm intra . (a) Autoradiographs of Western blots showing that nd 3 embryos reared at 25°C overproduce a form of N resembling NΔCterm (lanes 1–3). This form of N, just like NΔCterm, is recognized by αNI and αNT (lanes 3 and 7) but not αNPCR (lane 6). The same amount of total proteins (quantitated by absorbance and BioRad D C protein assay kit) was loaded in all of the lanes (note similar levels of NFull in all lanes). 0–3-h (25°C) or 0–6-h (18°C) embryos were used. The blot probed with αNI (lanes 1–3) was reprobed with αNPCR (lanes 4–6). Lane 7 is an independent blot. The levels of NFull and NΔCterm in Canton S embryos at 18°C were similar to the levels observed at 25°C (data not shown). 4% gel was used because in these gels NFull and N350.2 migrate together resulting in unambiguous determination of the level of NΔCterm. All N forms shown migrate in the ∼350-kD range. (b) Autoradiographs of Western blots showing that nd 3 embryos reared at 25°C also overproduce forms of N resembling NΔCterm TMintra and NΔCterm intra . Lanes 4–6 are from the same blot but lanes 2 and 3 are exposed to film for a shorter period than lane 1. Lanes 1–3 are an independent blot. S2-N in lane 1 was treated for 1 h, whereas S2-N cells in lane 6 were treated for 2 h. The different ∼350-kD forms of N do not resolve in 8% gels used for this blot. W-Ab, Western blotting antibody; Temp, temperature at which embryos were reared after heat-shock.

Article Snippet: The amount of proteins in different extracts was standardized using absorbance values at 280 nM and the BioRad D C protein assay kit.

Techniques: Western Blot