Ab96379

Ovarian cancer cells were disrupted using cell lysis buffer (Promega) on ice for 30 min and centrifuged at 12000 rpm for 30 min. The supernatant was transferred into the new centrifuge tube, and the concentration of protein samples was detected using the BCA-200 Protein Assay kit (Pierce, Rockford, IL, USA). Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore). The non-specific sites in the membrane were blocked using 5% skim milk for 1 h, followed by incubation with primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibody. The blots were visualized using the enhanced chemiluminescent (ECL) system (Beyotime, Shanghai, China). The primary antibodies, including phosphorylated extracellular regulated MAP kinase (p-ERK; ab214036), ERK (ab17942), p-MAP kinse-ERK kinase (p-MEK; ab96379), MEK (ab178876) and GAPDH (ab181602) were purchased from Abcam (Cambridge, MA, USA).

The paraffin-free tissue sections were treated with citrate antigen repair buffer (pH 6.0), and then the tissue sections were incubated with peroxidase blocking solution (S2023, Dako) for 15 min and blocked with protein (X0909, Dako) for 30 min. The corresponding specific primary antibodies were used: phosphor-ser221-MEK (rabbit, ab96379, Abcam), phospho-ERK1/2-Thr202/Tyr204 (rabbit, 4370, CST) and phospho-ser473-AKT (rabbit, 4060, CST). After treatment, the slides were incubated overnight at 4°C. Rabbit HRP conjugated secondary antibody (K4003, Dako) and hematoxylin (MHS32, Sigma) counterstain were used for treatment. The samples were observed with a digital microscope (Panoramic Viewer, 3D HISTECH), and protein expression was measured with a histochemical score (H-Score). Immunohistochemical results were scored with H-SCORE. The number of positive cells in each section H-SCORE = ∑ (PI × I) = (percentage of weak-strength cells × 1) + (percentage of medium-strength cells × 2) + percentage of cells. In this formula, PI represents the percentage of positive cells in the section, and I represents the staining intensity (9 –11 (link)).

After lysing in RIPA buffer, the collected total protein was separated on 12% SDS-PAGE and shifted onto PVDF membranes. 5% nonfat milk powder was added for blocking membranes. Next, the membrane was probed all night at 4 ​°C with primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA), TRAP (ab65854, 1/1000; Abcam), NFATc1 (ab175134, 1/1000; Abcam), TRAF3 (ab36988, 1/1000; Abcam), TRAF1 (ab129279, 1/1000; Abcam), TRADD (ab110644, 1/1000; Abcam), TRAF2 (ab230795, 1/1000; Abcam), ADAM17 (ab39162, 1/1000; Abcam), TNFRII (ab8161, 1/1000; Abcam), TNFRSF1A (ab19139, 1/1000; Abcam), p-IKB (ab133462, 1/1000; Abcam), IKB (ab32518, 1/1000; Abcam), TNFRSF11 (NB100-56508, 1/1000; Novus Biologicals, Littleton, CO, USA), p-IKKα (ab38515, 1/1000; Abcam), IKKα (ab32041, 1/1000; Abcam), p-IKKβ (ab59195, 1/1000; Abcam), IKKβ (ab124957, 1/1000; Abcam), p-Erk (ab214036, 1/1000; Abcam), Erk (ab184699, 1/10000; Abcam), p-MEK (ab96379, 1/1000; Abcam), MEK (ab32091, 1/1000; Abcam). On the following day, the HRP-secondary antibodies were added for 2 ​h at room temperature. The protein density was examined by the ECL luminous liquid (Pierce, Rockford, IL, USA). The assay was taken in triplicate.

Xenograft tumours and human lung tissues were fixed in formalin, embedded in paraffin, and sectioned into 5 μm thickness after embedding. For H&E staining, the sections were double stained with haematoxylin and eosin, and histopathological changes were analysed under a light microscope.
For immunohistochemistry, the sections were blocked with 5% goat serum (ZLI-9021, ZSGB-BIO, Beijing, China) for 60 min and then incubated with the primary antibodies against IMP4 (ab181046; Abcam), ki67 (ab16667; Abcam), p-MEK1 (ab96379; Abcam), and p-ERK (ab201015; Abcam) at 4°C overnight. After incubation with HRP-conjugated secondary antibody for 60 min, the sections were stained with 3′, 3-diaminobenzidine (DAB; ZLI-9017, ZSGB-BIO). Finally, the slides were observed and photographed under a light microscope.

The total proteins from lymphocytes were isolated by RIPA (Biorbyt, orb402072) protein lysate, separated by 10% SDS-PAGE gel electrophoresis, and transferred onto a membrane for 90 min. Then, the membrane was blocked at room temperature for 1 h using 1% BSA (Solarbio, PC0001), primary antibody dilutions MEK (Abcam, ab32091), ERK1/2 (Abcam, ab17942), p-MEK (Abcam, ab96379), p-ERK1 (Abcam, ab200807), p-ERK2 (Abcam, ab151549), PI3K (Abcam, ab106693), Akt (Abcam, ab2732), mTOR (Abcam, ab182651), p-Akt (Abcam, ab192623), p-mTOR (Abcam, ab137133), and β-actin (Abcam, ab8227) were subsequently added and incubated at room temperature for 2 h. After washing the membrane three times with TBST buffer, the cells were added with HRP-labeled secondary antibody (Abcam, ab205718) and incubated at room temperature for 0.5 h, then washed with TBST buffer for three times. The protein bands were developed by ECL luminescence kit (Absin, abs920) for detection.