Ab194754

HCC tissues and cell lines were lysed using M-PER mammalian protein extraction reagent (Pierce, Thermo Fisher Scientific, Inc.) containing the protease inhibitors following the standard procedures. Protein samples (40 μg) were separated in 4–12% SDS–PAGE gel and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, San Francisco, CA). Western blotting was performed according to the manufacturer’s protocols. The following antibodies purchased from Abcam were used in this study: Syncytin-1 (1:2000, ab179693), MEK1/2 (1:5000, ab178876), p-MEK1/2 (S218 + S222) (1:1000, ab194754), c-fos (1:2000, ab208942), c-myc (1:1000, ab32072), c-jun (1:1000, ab31419), CCND1 (1:2000, ab40754), CDK4 (1:1000, ab108357), rabbit IgG (1:2000, ab6721), and β-actin (1:5000, ab227387). ERK1/2 (1:1000, A10613), and p-ERK1 (T202/Y204)/ERK2 (T185/Y187) (1:1000, AP0472) were from ABclonal Technology. β-actin was used as an internal control. The immunoreactive bands were visualized using an ECL reagent (Pierce) according to the manufacturer’s recommendation. The bands were quantified by densitometry with ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA).

The protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA). The membrane was incubated with antibodies against human LC3B (ab192890, Abcam), Beclin-1 (#4122, Cell Signaling Technologies), MEK1/2 (ab178876, Abcam), phosphorylated (p)-MEK1/2 (ab194754, Abcam), Nrf-2 (#12721, Cell Signaling Technologies), and GAPDH (KC-5G4, Aksomics, Shanghai, China). Goat anti-mouse IgG (H + L) (KC-5G4, Southern Biotech, Birmingham, AL, USA) coupled with peroxidase was used as the second antibody. The intensity of staining was visualized using an X-ray image film processor (Kodak, Rochester, NY, USA).

Cells were collected by trypsin detachment and lysed using Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing the protease inhibitors (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China). The concentration of protein was determined using the bicinchoninic acid (BCA) kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China). The protein was then separated by conducting sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. A membrane blockade was performed for 1 h and incubated with the following primary rabbit anti-human antibodies to matrix metalloprotein-9 (MMP-9) (ab194316, 1 : 1000), N-cadherin (ab18203, 1 : 1000), Vimentin (ab137321, 1 : 500), E-cadherin (ab15148, 1 : 500), ERK1/2 (ab17942, 1 : 1000), phosphorylated ERK1/2 (ab214362, 1 : 500), MEK1/2 (ab178876, 1 : 5000, Abcam, Cambridge, UK), phosphorylated MEK1/2 (ab194754, 1 : 1000), and GAPDH (ab181602, 1 : 5000) at 4°C overnight. The aforementioned antibodies were provided by Abcam Inc. (Cambridge, UK). The protein bands were developed using the enhanced chemiluminescence reagent and the observations were documented using the SmartView Pro 2000 software (UVCI-2100, Major Science, USA). The Quantity One software analyzed the gray values of protein bands.