Rabbit polyclonal anti s100a4

Taking into account the normalisation factors derived from the reference proteins, 10 ug of protein from tissue lysates of normal, FA, FTC and PTC samples were separated on 4-12% Bis-Tris NuPAGE gels and electroblotted onto nitrocellulose (Bio-Rad). The membrane was blocked with 5% skim milk in TBS for 1 h at RT. For S100A4 analysis, the membrane was cut in three sections from around 5-17 kDa, 17-32 kDa and >32 kDa. The sections were incubated overnight at 4°C with a) 5-17 kDa - rabbit polyclonal anti-S100A4 (1:100, AbCam), b) 17-32 kDa - rabbit monoclonal anti-Rab7a (1:500, Cell signaling) and c) rabbit monoclonal anti-β-Tubulin (1:500, Cell signaling). For S100A13 analysis, the membrane was cut in two sections (5-17 kDa and >32 kDa) and incubated overnight with either rabbit monoclonal anti-S100A13 (1:3000, AbCam) or rabbit monoclonal anti-Rab7a (1:500, Cell signaling). The secondary antibody was fluorescence-labelled IRDye 800CW goat anti-rabbit IgG (H + L) (1:10 000 in 5% skim milk in TBST, LI-COR), incubated at RT for 1 h and visualized with the Odyssey infrared imaging system.

Cultured cells were harvested and lysed in ice-cold RIPA buffer and equal quantities of total protein were separated by 10% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes (Protran, Whatman, Dassel, Germany) and blots were probed with the indicated primary antibodies, as previously described (Cortes-Canteli et al., 2004). The antibodies used were the following: rabbit polyclonal anti-C/EBPβ, rabbit polyclonal anti-Oct3/4 (Santa Cruz Biotechnology, CA), rabbit polyclonal anti-S100A4 (Abcam), rabbit polyclonal anti-Musashi-1 (Abcam) and mouse monoclonal anti-α-tubulin (Sigma). Secondary peroxidase-conjugated donkey anti-rabbit and rabbit anti-mouse antibodies were from Amersham Biosciences (GE Healthcare, Buckinghamshire, England) and Jackson Immunoresearch, respectively. The Western blots shown in Figures 1, 3, 5 and 8 are representative of at least three independent experiments.