Anti mek1 2

Cells or tissues were lysed in RIPA buffer, including a protein-phosphatase inhibitor cocktail (Thermo Scientific, USA) at 4℃, then use 10% SDS-PAGE gels to separate the total proteins before they are transferred electrophoretically (Bio-Rad, USA) onto PVDF membranes (Millipore, USA). Bio-Rad ChemiDoc XRS +System was used to detect the protein after dropping the developer solution (Bio-Rad, USA) onto the blots. The primary antibodies were purchased in this study as follows: anti-LAMC1 (PAB32791; 1:1000; Bioswamp, China), anti- CEBPα (18311-1-AP; 1:1000; Proteintech, USA), anti- PPARγ (16643-1-AP; 1:1000; Proteintech, USA), anti-DLK1 (10636-1-AP; 1:1000; Proteintech, USA), anti-HSL (17333-1-AP; 1:1000; Proteintech, USA), anti-Perilipin-1 (9349; 1:1000; Cell Signaling, USA), anti-MEK1/2 (11049-1-AP; 1:1000; Proteintech, USA), anti-p-MEK1/2 (phosphor Ser217/221) (3958; 1:1000; Cell Signaling, USA), anti-ERK1/2 (16433-1-AP; 1:1000; Proteintech, USA), anti-p-ERK1/2 (phosphor Thr202/Tyr204) (AF1015; 1:1000; Affinity Biosciences, USA), anti-STAT3 (10253-2-AP; 1:1000; Proteintech, USA), anti-p-STAT3 (phosphor Tyr705) (9145; 1:1000; Cell Signaling, USA), mTOR (2983T; 1:1000; Cell Signaling, USA), anti-p-mTOR (phosphor Ser2448) (5536T; 2983T; 1:1000; Cell Signaling, USA), anti-GAPDH (sc-32233; 1:5000; Santa Cruz, CA), and anti-β-tubulin (sc-5274; Santa Cruz, CA).

We first separated proteins from tissues or cells using RIPA lysis buffer (Merck Millipore, Waltham, MA, USA) and then assayed the corresponding protein concentrations employing BCA kits (Solarbio Co.). After electrophoresis of the samples, we transferred them to polyvinylidene difluoride (PVDF) membranes supplied by Merck Millipore. These membranes were then incubated for 2 h in a solution containing 5% skim milk. After adding the primary antibody solution to the samples, they were incubated overnight at 4°C. After a 2-h incubation with the secondary antibody, the membranes were washed three times with TBST. The bands were then observed. We used the imaging system from Bio-Rad Laboratories (Hercules, CA, USA). The following antibodies were used: anti-PYGB (1:1000, Proteintech, #12075), anti-MEK1/2 (1:5000, Proteintech, #11049), anti-ERK1/2 (1:2000, Proteintech, #11257), anti-P-ERK1/2 (1:1000, Proteintech, #28733), anti-P-MEK1/2 (1:1000, Cell Signaling Technology, #9154), anti-α-Tubulin (1:2000, Proteintech, #66031), HRP-goat anti-rabbit IgG (1:2000, Proteintech, #SA00001-2), and HRP-goat anti-mouse IgG (1:2000, Proteintech, #SA00001-1).