Ab180772

Panc-1 cells in the logarithmic growth phase were first collected and digested with 0.25% trypsin. Next, radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Shanghai, China) was applied to extract total protein. A bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was employed for the quantification of protein concentrations and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad) was utilized for protein separation. After that, polyvinylidene fluoride (PVDF) membranes (Invitrogen) were employed for protein transference following the producer’s guidelines and sealed in 5% skim milk at 37°C for 2 h. The membranes were placed into a plastic bag in the next step and incubated overnight with primary antibodies against KRAS (ab180772, 1 : 500, Abcam, Cambridge, MA, USA) and GAPDH antibody (ab9485, 1 : 2000, Abcam) at 4°C. After 3 times 5-min washing with phosphate buffered saline (PBS), at room temperature, 1.5-h incubation of membranes with secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG H&L (1 : 2000) was conducted, followed by again washing 3 times for 5 min with PBS. An electrochemiluminescent (ECL) detection system (Thermo Scientific, MA, USA) was employed for signal detection. The density of protein bands was quantified or analyzed using Quantity One v4.6.2 software (Bio-Rad).

20 μg of protein was added to SDS-PAGE as quantified. After protein separation, the membrane was transferred using activated PVDF membrane (Thermo Fisher, 22,860). The membrane was incubated with specific antibodies. Subsequently, the PVDF membrane was incubated with the HRP secondary antibody at room temperature. The antibodies used were as follows: Ras (abcam, ab180772), Raf (abcam, ab200653), Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, 9101S), p-ERK1/2 (Thr202, Tyr204) (Thermo Fisher, 14–9109-82) and GAPDH (Cell Signaling Technology, 2118). They were diluted according to the manufacturer’s instructions.

The extraction of proteins from tissues or cells was implemented via radioimmunoprecipitation assay (RIPA, Sangon Biotech, Shanghai, China). Then, the protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (ThermoFisher, Waltham, USA). After the block by 5% non-fat milk, Western blot analysis was applied to determine the protein expression [14 (link)]. The membranes were incubated with the diluted primary antibodies (1:2000) and secondary antibodies (1:5000), respectively. Finally, the protein signals were achieved using an enhanced chemiluminescence (Amersham Pharmacia, Piscataway, USA). Primary antibodies against Ras (1:1,000, ab180772), phosphor-ERK1/2 (p-ERK1/2, 1:1,000, ab47339), ERK1/2 (t-ERK1/2, 1:3,000, ab196883), PCNA (1:2,000, ab18197), OCT4 (1:2,000, ab109183), hexokinase 2 (HK2) (1:2,000, ab227198), HMGA2 (1:2,000, ab97276), β-actin (1:2,000, ab8227), GAPDH (1:2,500, ab9485) and secondary antibodies (1:2,000, ab205718) were all obtained from Abcam. ImageJ software was used to evaluate the band density by densitometric analysis.