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. 2012 Apr 13;287(24):20356–20368. doi: 10.1074/jbc.M112.349738

FIGURE 5.

FIGURE 5.

Elimination of Notch1 and Notch2 proteins in primary cortical neuronal cultures after Cre transduction. Shown is the time course of Notch1 (A) or Notch2 (B) inactivation in fNotch1/fNotch1;fNotch2/fNotch2 cortical neuronal cultures after Cre transduction. The lentivirus carrying the cDNA encoding either a GFP/defective Cre (Cre−) or a GFP/functional Cre fusion protein (Cre+) was introduced to infect cortical neuronal cultures derived from fNotch1/fNotch1;fNotch2/fNotch2 neonate pups at DIV1. Total cell lysates were collected at the designated days (DIV2–10) and were analyzed (10 μg of lysates per lane) by Western blotting using specific antibodies for the Notch1 (rabbit polyclonal, from A. Israel) or the Notch2 (rat monoclonal, clone C651.6DbHN from the Hybridoma Bank, University of Iowa) C-terminal domain. α-Tubulin or VCP (Vasolin containing protein) was used as a loading control. Although most Notch proteins were subjected to S1 cleavage (Cleaved, ∼95 kDa), full-length (FL) Notch (∼270 kDa) could be easily detected in this culture system. In the presence of Cre, inactivation of Notch1 and Notch2 occurs rapidly, and loss of Notch1 and Notch2 proteins is complete by DIV4.