Western Blot Analysis of Notch Signaling

Whole cell lysates were prepared as described ^{13 (link), 36 (link)}. 20 µg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Invitrogen). Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for Notch1 (1:1000 rat monoclonal 5B5; Cell Signaling, Danvers, MA), ICN1^Val1744 (1:1000 rabbit monoclonal anticleaved NOTCH1 Val1744 D3B8; Cell Signaling), ICN3 (1:1000 rat monoclonal anti-NOTCH3 8G5; Cell Signaling), JAG1 (1:1000 rabbit monoclonal anti-Jagged1 28H8; Cell Signaling), pRb (1:1000 rabbit polyclonal anti-Phospho-Rb Ser⁷⁸⁰; Cell Signaling), p16 (1:1000 mouse monoclonal anti-Human p16 G175–1239; BD Biosciences), p15 (1:200 mouse monoclonal anti-p15 15P06; Santa Cruz), p21 (1:1000 mouse monoclonal anti-Human Cip1; BD Biosciences), p53 (1:1000 Rabbit polyclonal anti-p53; Cell Signaling) IVL (1:1000 mouse monoclonal anti-Involucrin clone SY5; Sigma-Aldrich, St Louis, MO), β-actin (1:30,000 mouse monoclonal anti-β-actin AC-74; Sigma Aldrich), Cat# A5316, and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). β-actin served as a loading control.