Donkey anti rabbit igg fitc

50% confluent MDA-MB 231 cells grown on 6 well plates were serum-deprived and then treated for 1 h with E2 and G1 alone or in the presence of GPER antagonist G-15 or VS-4718 FAK inhibitor, as indicated. Next, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with or without (negative control) a rabbit primary antibody against STAT3 (Cell Signaling Technology, Milan, Italy). After incubation, the wells were extensively washed with PBS and incubated with donkey anti-rabbit IgG-FITC (1:400; purchased from Alexa Fluor, Life Technologies, Milan, Italy) for 1 h at room temperature. Finally, cells were washed with PBS and incubated in PBS buffer containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), (1:1000), (Sigma-Aldrich, Milan, Italy) 10 min at room temperature for nuclear staining. Imaging showing nuclear STAT3 accumulation were acquired on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT) and analysed using the software Gen5 (BioTek, Winooski, VT).

5 × 10⁴ MDA-MB 231 and SUM159 cells were grown on 6 well plates. When reached 50% confluence, cells were serum-deprived for 12 h and then treated for 30 min with IGF-1. Next, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with or without (negative control) a rabbit primary antibody against Y397-FAK (Cell Signaling Technology, Milan, Italy). After incubation, the wells were extensively washed with PBS and incubated with donkey anti-rabbit IgG-FITC (1:400; purchased from Alexa Fluor, Life Technologies, Milan, Italy) for 1 h at room temperature. Finally, cells were washed with PBS and incubated in PBS buffer containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000; Sigma-Aldrich, Milan, Italy) 10 min at room temperature for nuclear staining. Images showing focal adhesion points among the cells were acquired on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT, USA) and analyzed using the software Gen5 (BioTek). The same procedure was also applied for FAK (Cell Signaling Technology, Milan, Italy) staining in MDA-MB 231 PTK2 WT and KO as well as in SUM159 PTK2 WT and KO. A total of 10 images for each condition was detected on the Cytation 3 Cell Imaging Multimode Reader (BioTek) and analyzed using the software Gen5 (BioTek).

MDA-MB 231 cells cultured on fibronectin-coated 6 well plates were serum deprived and then treated for 30 min with E2 and G1 alone or in combination with G15, as indicated. Then cells were washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with or without (negative control) a rabbit primary antibody anti p-FAK (Y397) (Cell Signaling Technology, Milan, Italy). After incubation, the wells were extensively washed with PBS and incubated with donkey anti-rabbit IgG-FITC (1:300; purchased from Alexa Fluor, Life Technologies, Milan, Italy) for 1 h at room temperature. Finally, cells were washed with PBS and incubated in PBS buffer containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), (1:1000), (Sigma-Aldrich, Milan, Italy) 10 min at room temperature for nuclear staining. FAs images were acquired on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT) and analysed using the software Gen5 (BioTek, Winooski, VT).