Anti p fak antibody

Immunoblotting was performed with rabbit anti‐ITGA3 antibody (1:250, HPA008572; SIGMA‐ALDRICH, St. Louis, MO, USA), anti‐Akt antibody (1:1000, #4691; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐Akt antibody (1:1000, #4060; Cell Signaling Technology), anti‐Erk1/2 antibody (1:1000, #4695; Cell Signaling Technology) and anti‐p‐Erk1/2 antibody (1:2000, #4370; Cell Signaling Technology), anti‐FAK antibody (1:1000, #3285; Cell Signaling Technology) and anti‐p‐FAK antibody (1:1000, #8556; Cell Signaling Technology). Anti‐GAPDH antibodies (1:1000, ab8245; Abcam, Cambridge, UK) were used as an internal control. The procedures were performed as described in our previous studies.15, 16, 22

Cells were harvested 48 h after transfection, and lysates were prepared. Immunoblotting was carried out with rabbit anti‐SKA1 antibodies (1:1,000 dilution, SAB2701430; Sigma‐Aldrich), anti‐AKT antibody (1:1000, #4691; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐AKT antibody (1:1000, #4060; Cell Signaling Technology), anti‐ERK1/2 antibody (1:1000, #4695; Cell Signaling Technology), anti‐p‐ERK1/2 antibody (1:2000, #4370; Cell Signaling Technology), anti‐FAK antibody (1:1000, #3285; Cell Signaling Technology) and anti‐p‐FAK antibody (1:1000, #8556; Cell Signaling Technology), anti‐SRC antibody (1:1000, #2123; Cell Signaling Technology) and anti‐p‐SRC antibody (1:1000, #6943; Cell Signaling Technology). Anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibodies (1:10000, ab8245; Abcam, Cambridge, UK) were used as an internal loading control. Experimental procedures were carried out as described previously.22, 25, 26, 27, 28 Protein expression was quantified by using NIH‐ImageJ.

After culture on a microscope cover glass (Thermo Scientific), the cells were treated with He, Ar, and N₂ of NTP, and incubated for an additional 24 h. The cells were fixed with 4% formaldehyde and blocked in bovine serum albumin in 5% PBS for 45 min. Slides were then incubated with a polyclonal rabbit anti-p-FAK antibody (1:50; Cell Signaling Technology) for 2 h, washed with PBS and incubated with an Alexa 546-labeled goat anti-rabbit antibody (1:250; Molecular Probe, Eugene, Oregon, CA, USA) for 45 min. After rinsing in PBS, Hoechst 33258 (Molecular Probe) was added to slides for 15 min to counterstain nuclei. Slides were washed with PBS, mounted with Vectashield (Vector laboratories, Inc., Burlingame, CA, USA), and then visualized using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).