Ab61201

Rat brain samples were homogenized in a lysis buffer (50mM Tris-HCl pH 7.4, 1mM EGTA; 150mM NaCl; 0.5% NP-40; 1mM NaF; 1mM sodium orthovanadate; 0.2% sodium deoxycholate; 1mM PMSF) containing protease and the phosphatase inhibitor. After centrifugation at 20,000g at 4 °C for 15 min, the supernatants were collected. Protein concentration was measured using Bradford method, by means of a spectrophotometer (Eppendorf). Then, 50μg of protein was mixed with a Laemmli sample buffer (5% 2-mercaptoethanol) and heated at 95 °C for 5 min. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (0.22 µm). Blots were probed with antibodies to HIF1α (1:1000, rabbit monoclonal, Abcam, ab179483), MKP1 (1:500, rabbit polyclonal, Abcam, ab61201), kRAS (1:500, rabbit polyclonal, Abcam, ab196630) and α-tubulin (1:8000, Sigma, T5168) diluted in tris buffered saline (TBS-T) 5% bovine serum albumin (BSA) overnight (4 °C). Then, signals were detected using horseradish peroxidase-conjugated secondary antibody (1:2000; mouse and rabbit Amersham; 60 min at room temperature in TBS-T 5% BSA) and an enhanced luminescence kit (ImmunoCruz, Santa Cruz Biotechnology).

After dewaxing, the paraffin sections with a thickness of about 4 μm were placed in a sodium citrate solution with pH = 7.0 heated in a microwaves on high heat for antigen retrieval. A 3% hydrogen peroxide solution was then dropped on the sliced tissue to block endogenous peroxidase. We then performed primary antibody incubation (anti-DUSP1, Abcam, ab61201, 1:100 dilution; anti-FOSB, Abcam, ab11959, 1:100 dilution), HRP goat anti-rabbit secondary antibody (1:200 dilution) incubation, and nucleus staining incubation. These were followed by DAB coloration, hematoxylin staining, and hydration, at which point we covered the slip with neutral resin and observed the final product under a microscope. We then used the Image J software to perform a semi-quantitative analysis.