Mouse anti cyclin e

The antibodies used included mouse anti-β-actin (#A5441; Sigma-Aldrich; 1:5000 for Western blot), mouse anti-HA (#AE008; ABclonal, Cambridge, MA, USA; 1:2000 for Western blot), rabbit anti-ubiquitin-like 3 (UBL3) (#A4028; Abclonal; 1:1000 for Western blot and 1:100 for immunohistochemistry (IHC)), rabbit anti-Ki67 (#ab15580; Abcam, Cambridge, MA, USA; 1:400 for IHC), rabbit anti-p27 (#sc-528; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000 for Western blot), rabbit anti-Cyclin D1 (#2922; Cell Signaling Technology, Beverly, MA, USA; 1:1000 for Western blot), and mouse anti-Cyclin E (#4129; Cell Signaling Technology; 1:1000 for Western blot).

Whole lysates of cells were prepared as previously described. Western blotting was performed with rabbit-anti-cyclin D2 (Cell Signaling Technology), mouse-anti-cyclin E (Cell Signaling Technology), rabbit-cyclin A2 (NOVUS Biologicals), mouse-cyclin D3 (Cell Signaling Technology), mouse-anti-β actin (Santa Cruz Biotechnology), rabbit-anti-ErbB-1 (Santa Cruz Biotechnology), rabbit-anti-ErbB-2 (Santa Cruz Biotechnology), rabbit-anti-ErbB-3 (Santa Cruz Biotechnology), mouse-anti-ErbB-4 (Santa Cruz Biotechnology), mouse-anti-phospho-Tyr (Millipore), rabbit-anti-phospho-Erk1/2 (Cell Signaling Technology), rabbit-anti-Erk1/2 (Cell Signaling Technology), rabbit-anti-phospho-Akt (Ser473) (Cell Signaling Technology), rabbit-anti-Akt (Cell Signaling Technology), rabbit-anti-phospho-mTOR (Cell Signaling Technology), rabbit-anti-mTOR (Cell Signaling Technology), or mouse-anti-GAPDH (Santa Cruz Biotechnology).

Immunostaining and confocal microscopy performed as described previously on a Zeiss 510 inverted confocal microscope28 (link). Primary antibodies include mouse anti-β-Gal 1:1000 (Promega); rabbit anti-LC8 (1:100) (W. Sale), mouse anti-Ci (1:50), mouse-α-Notch 1:10, mouse anti-Wg (1:800), mouse anti-Cut (1:100) and mouse anti-DIAP1 (1:50) (DSHB); rabbit α-yki 1:1000 (K. Irvine); mouse α-rat CD2 1:100 (Research Diagnostics, Inc.); mouse anti-V5 (1:200, Invitrogen); mouse anti-dpERK (1:10,000, Sigma); rabbit anti-phospho histone H3 (1:100), rabbit anti-phospho-Smad1/5 (1:100), rabbit anti-DCP1 (1:150), rabbit anti-Caspase3 (1:100) (Cell Signaling); mouse anti-dMyc (monoclonal P4C4-b10); mouse anti-Cyclin E (H. Richardson). BrdU assays performed as described previously (Robinson et al. 2010) with mouse anti-BrdU (1:100; Becton Dickinson).