Ep715y

IHC analysis was performed as we previously described^{40 (link)}. The rabbit polyclonal antibody against CDK5 (EP715Y, Abcam) was used for detection. For evaluating expression of CDK5 in LUAD tissues, the staining was visualized and classified based on the percentage of positive cells and the intensity of staining according to the 0–4 semi-quantitative system described in our previous study^{40 (link)}. The total scores were determined by multiplying the percentage score and intensity score and graded as low for score of 0–4 and high for score of 5–12. Each section was scored independently by two pathologists and a third pathologist determined the final score, if there was any inconsistency.

Cells were lysed in lysis buffer, and protein concentrations were measured with the BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated by 10% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated in PBS containing 5% bovine serum albumin for 2 h at room temperature, followed by overnight incubation at 4 °C with different dilutions of the primary antibodies, including antibodies to CDK5 (EP715Y, Abcam, Cambridge, MA, USA), ZEB1 (ab181451, Abcam), E-cadherin (4A2, Cell Signaling Technology, Boston, MA, USA), N-cadherin (D4R1H, Cell Signaling Technology), Twist (ab50581, Abcam), Snail (C15D3, Cell Signaling Technology) and GAPDH (ab9485, Abcam). The membranes were developed with the Odyssey infrared imaging system according to the manufacturer’s instructions. The levels of protein in each sample were normalized relative to those of GAPDH. Each experiment contained triplicate wells of each sample, and all experiments were repeated at least three times.