Notch Pathway Profiling by RT2-PCR

The Human Notch Pathway RT²-Profiler™ PCR Array (#PAHS-059, Invitrogene) was conducted as described [9 (link)]. All samples were normalized to the levels of β-actin, and results are expressed as relative fluorescence intensity. 4%-20% gradient SDS-PAGE gel was used for Hes-1 in Fig 2C-related experiment, while 10% SDS-PAGE gels were used for other Western blot experiments. Immunoblot was performed as described [17 (link)]. Membranes were probed with Abs to Notch1 (ab52627, Abcam for Fig 2C; ab27526, Abcam for Fig 2D; and N4788, Sigma-Aldrich for Fig 3A, respectively), Notch4 (TA321658, Origene) that detected full length Notch4 expression in MAFs, activated Notch1 (for Fig 3A, N4788, Sigma-Aldrich), Hes-1 (ab71559, Abcam for Fig 2D and Fig 3A; ab55265, Abcam for Fig 2C, respectively), Hey-1 (GTX42614, GeneTex), WISP-1 (SAB1400324, Sigma-Aldrich) or β-actin (A1978, Sigma-Aldrich) accordingly. Autophotographs of blots were scanned by densitometer (Molecular Dynamics) to quantify the bands.