Ab176660

Lysed in RIPA buffer, CC cells were subjected to protein quantification via a bicinchoninic acid assay. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was applied for the separation of proteins, followed by transferring to polyvinylidene difluoride membranes. After blocking, the membrane was incubated with primary antibodies. Further, the horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) were supplemented. The enhanced chemiluminescence system (Thermo Fisher Scientific) was employed for evaluating the protein bands, and the proteins were quantified via Image J software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used in this study included anti-FLOT2 (1:3000, ab96507, Abcam), Vimentin (1:1000, ab16700, Abcam), N-cadherin (1/1000, ab245117, Abcam), E-cadherin (1 µg/ml, ab231303, Abcam), MEK (1:10000, ab32576, Abcam), p-MEK (1 µg/ml, ab278716, Abcam), ERK1/2 (1/10000, ab184699 Abcam), and p-ERK1/2 (0.2 ng/ml, ab176660, Abcam).

Cells were seeded onto poly-d-lysine-coated coverslips. Then, methanol-free 4% paraformaldehyde was used to fix cells for 15 minutes at room temperature. Subsequently, samples were blocked with 5% goat serum at room temperature for 45 minutes. After washing with TBST, samples were incubated with primary antibody at 4°C overnight. The primary antibodies used in the present study were as follows: p-ERK (1:1,000, #ab176660), Yap (1:1,000, #14074), Tom20 (1:1,000, #ab186735), and cyt-c (1:1,000, #ab90529; Abcam). Subsequently, samples were rinsed three times with TBST for 15 minutes and followed by incubation with secondary antibody for 45 minutes at room temperature; after rinsing three times for 5 minutes using TBST, the samples were labeled with DAPI to tag the nuclei. Cells were observed using a laser confocal microscope (TcS SP5).27 (link)

Harvested cells (2×10⁶) were placed into 1.5 mL Eppendorf tubes and mixed with cell lysate [99 µL cell lysate + 1 µL phenylmethylsulfonyl fluoride (PMSF)] and incubated on ice for 30 min. They were then centrifuged at 3,500 rpm for 30 min at 4 °C, the liquid (total protein) was carefully removed into another EP tube. Western blot was performed using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separations, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% skimmed milk at 37 °C for 1 h. Membranes were incubated 1:1,000 dilutions of the following primary antibodies: anti-ERK (ab32537), anti-p-ERK (ab176660), anti-p38 MAPK (ab197348), anti-p-p38 MAPK (ab176664), and anti-microtubule protein (ab11304) (Abcam, USA). Then, the membranes were incubated with a 1:2,000 dilution of anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to detect the protein expression.