Fak primary antibody

The fluorescence labeling procedure of HSkM, RH-30 and HA-OH1 cells was performed as described previously [32 (link), 40 (link)]. Briefly, the cells were grown in Ibidi dishes/ slides (Ibidi GmbH, Martinsried, Germany), fixed in 4% paraformaldehyde (Santa Cruz, Dallas, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labeled with anti-SGPL1 primary antibody ((H-300) #sc-67368, Santa Cruz, USA) and Alexa Fluor 488 dye secondary antibody (Thermo Fisher Scientific Inc., USA). The co-localization experiments were performed by additional labeling with F-Actin antibody Phalloidin-Alexa 596 (Invitrogen, USA), focal adhesion kinase (FAK) primary antibody (#3285, Cell Signaling, USA) or with cell-permanent ER-Tracker™ Green dye (BODIPY® FL glibenclamide, Molecular Probes, USA). All samples were also counter-stained with Hoechst (PanReacAppliChem, Darmstadt, Germany). Images concerning on SGPL1 expression and localization were captured on a confocal laser-scanning microscope Leica DMi8 (Leica, Germany). Transfection efficiency (GFP signal of SGPL1; TYE-563 positive siRNA control) was controlled with a fluorescence microscope CKX53 (Olympus, Japan).

IHC staining was performed as previously described [35 (link)]. In brief, blocks of tissue samples embedded in paraffin were cut into 4-μm-thick sections, de-paraffinized and rehydrated. Antigen retrieval was achieved by autoclaving the sections at 121 °C for 10 min in a pH 6.0 antigen-retrieval solution (DAKO, Carpinteria, CA, USA). Endogenous peroxidase activity was blocked upon incubation in 3% hydrogen peroxide (Sigma) for 10 min. The sections were then incubated with the FAK primary antibody (Cell Signaling Technology) at room temperature for 1 h. The DAKO REAL™ EnVision™ Detection System EnVision (DAKO) was then applied for 1 h. Finally, the sections were incubated in 3′3-diaminobenzidine for 5 min, counterstained with Mayer’s hematoxylin and mounted. Negative controls were prepared by replacing the primary antibodies with non-immune serum.