Clx infrared imager

On-cell western [74 ] was performed with U2OS MYC-ER cells. 72,000 cells were plated in each well of a 24-well plate, and 24 hours later, treated ± 4OHT for 48 hours. Cells were then fixed with 3.7% paraformaldehyde (Sigma) but not permeabilized. Wells were washed with tris-buffered saline, and stained with primary antibodies mouse anti-LAT1 BU53 (Novus NBP2-50465AF647) or mouse IgG2A isotype control (Novus IC003R), and secondary antibodies goat anti-mouse Alexa Fluor 790 (Thermo Scientific A11357) or CellTag 700 Stain (Licor 926–41090), which is used to quantify total cell number and intensity. The stained plate was then digitally imaged using a Licor Odyssey CLx infrared imager (Licor), and well intensities in both channels (700 nM for CellTag, 800 for LAT1 or isotype control) were quantified using ImageStudio software (Licor). Individual wells were treated as biological replicates, and results are representative of two separate experiments.

CNE2 cells with different concentrations of luteolin (0 μM, 20 μM, and 40 μM) and CIS (10 μM) were incubated for 36 h, total protein was extracted and quantified, and 50 μg of protein was used for western blot analysis. The appropriate concentration of separating gel and stacking gel was chosen depending upon the molecular weight of the target protein. The samples were then loaded, onto the SDS-PAGE gel, electrophoresed and the resolved proteins were transferred to a transfer membrane (PVDF), and blocked. Diluted β-actin (Cell Signaling Technology, CST, MA, USA), p-ERK1/2 (CST), ERK1/2 (CST), AKT (CST), PI3K (CST), PCNA (Bioss, Woburn, MA, USA), and XIAP (CST) at a dilution ratio of 1 : 1,000 were incubated with the PVDF membrane overnight at 4°C, then wash three times with TBST, for 10 min each. Then, the corresponding diluted goat anti-mouse or goat anti-rabbit secondary antibody was added, and incubated with the PVDF membrane at room temperature for 2 h, washed with TBST three times for 10 min and then scanned on an ODYSSEY CLx Infrared Imager (LICOR, Lincoln, NE, USA) to detect the signal intensities of the immunoreactive bands. Using β-actin as an internal reference, the comparative protein expression levels could be calculated, the experiment was performed in triplicate.