Ab18056

Human OSCC cells were lysed by using RIPA lysis buffer containing proteinase inhibitors (Beyotime, China). The concentration of proteins was detected according to the instruction of the BCA Protein Quantitation kit (Beyotime, China). Then, the total proteins (60 µg) were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto a PVDF membrane (Millipore, USA). Then, 5% skim milk was applied to block the membranes at room temperature for 120 min. Subsequently, immunoblotting was performed with specific antibodies against NEDD9 (1:1000, ab18056, Abcam, Cambridge, UK) and β-actin (1:2000, β-actin, Abcam, Cambridge, UK). β-actin was used as an internal control. The membranes were incubated with primary antibodies at 4°C temperature overnight. Next day, the secondary antibodies (Abcam, Cambridge, UK) were added and incubated at room temperature for 2 h. Ultimately, the signals were detected by an enhanced chemiluminescence (ECL) detection system (PerkinElmer, USA) and quantified with ImageJ software.

Lung tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and deparaffinized before being incubated with NEDD9 antibody (1:200, #ab18056, Abcam), Anti-Histone H3 (acetyl K9) (1:200, #ab10812, Abcam) and Phospho-FAK (Tyr397) (1:200, # 44-624 G, Invitrogen) at 4 °C overnight. Secondary antibodies were then incubated at 37 °C for 30 min.
IHC was independently assessed by pathologists from ZuoCheng Biological Technology LTD, Shanghai, China. They assessed the expression of proteins using the quick score method, which considers both the proportion and intensity of stained cells. Briefly, the proportion of positively stained cells was measured on a scale of 1–6 (1:1–4%; 2: 5–19%; 3: 20–39%; 4: 40–59%; 5: 60–79%; and 6: 80–100%). The staining intensity of positive cells was scored from no staining 0 to strong staining 3. The immunohistochemistry score was calculated by multiplying the percentage score by the intensity score, ranging from value of 0 to 18.

Total protein was extracted in lysis buffer with protease inhibitors. After proteins were separated on a 10% SDS-PAGE gel, they were transferred to a cellulose acetate membrane. After blocking with 5% skim milk, the membrane was incubated with NEDD9 (1:1000, ab18056, Abcam, USA) and β-actin (1:10000, A5316, Sigma, USA) antibodies at 4 °C overnight. The membrane was subsequently washed and incubated with a goat anti-mouse secondary antibody (1:10,000, 115–035-003, Jackson, USA) for 2 h at room temperature. After the membrane was fully washed, chemiluminescence was performed for the development and visualization of the protein bands.