Rabbit monoclonal anti rab7a

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.

Immunobloting was performed as previously described23 (link). Briefly, 15 μg of protein were separated on 4–12% Bis-Tris NuPAGE gel and electroblotted onto nitrocellulose. The membrane was blocked with 5% skim milk, cut in two sections around 30 kDa and incubated overnight at 4 °C with either mouse monoclonal anti-decorin (1 μg/mL, clone 115402 R&D Systems) or rabbit monoclonal anti-Rab7a (1:500, Cell signalling). The secondary antibodies were fluorescence-labelled IRDye 800CW donkey anti-mouse and goat anti-rabbit IgG (LI-COR), incubated at RT for 1 h, visualised and quantitated using an Odyssey infrared imaging system and software(LI-COR, Lincoln, NE). Densitometry values were obtained using a fixed size rectangle feature enclosing the band of interest to obtain the intensity output after background subtraction (median intensity of pixels around the rectangle area). Measured values for decorin were divided by Rab7a values (reference control23 (link)) to compare decorin expression across samples.