Bca protein assay kit

Cells were homogenized in cold RIPA buffer with phenyl methane sulfonyl fluoride (PMSF) and phosphatase inhibitor. Protein concentrations were determined by the Bicinchoninic Acid (BCA) Protein Assay Kit (Sparkjade, Jinan, China). Equal protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, MA, United States). PVDF membranes were blocked with 5% skimmed milk powder and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study are as follows: DDIT4 (1:500; ER1706-76, HUABIO, Hangzhou, China), AKT1 (1:500; ER1609-47, HUABIO, Hangzhou, China), phosphorylated AKT1 (Ser473) (p-AKT1; 1:500; ER1607-73, HUABIO, Hangzhou, China), RP70S6KB1 (1:1,000; #2708, Cell Signaling Technology, Boston, United States), p-RP70S6KB1 (Thr389) (p-S6K; 1:1,000; #9234, Cell Signaling Technology, Boston, United States), Raptor (1:500; ER1802-57, HUABIO, Hangzhou, China), Rictor (1:500; EM1709-50, HUABIO, Hangzhou, China), and β-actin (1:1,000; R1207-1, HUABIO, Hangzhou, China). After staining with HRP-conjugated secondary antibody, protein bands were visualized using the Immobilon Western HRP Substrate Kit (Millipore, MA, United States).

Levels of inflammatory factors and neurotransmitters were measured by Enzyme-linked Immunosorbent Assay (ELISA) in serum and brain tissue, as well as in the colon. Centrifuge the whole blood sample collected in the serum separation tube at 3,000 × g for 15 min, and then stored the supernatant at −80°C. The tissues were raised with pre-cooled PBS (0.01M, pH = 7.4), and cut into pieces after weighing. Put the trimmed tissue into PBS solution (1:9 weight-volume ratio, and added protein inhibitor to PBS) for homogenization, finally centrifuge the homogenate at 5,000 × g for 10 min, and the supernatant was stored at −80°C. A bicinchoninic acid (BCA) Protein Assay kit (SparkJade, China) was used to determine the protein concentration. Levels of IL-6, IL-1β, IL-18 and TNF-α were determined using ELISA Assay kits (JiangLaibio, China) according to manufacturer's instructions. The concentration of 5-HT, DA, NE, HIAA, HVA and DOPAC were determined using the ELISA Assay kits purchased from Jiangsu Meimian Industrial Co, Ltd. (Jiangsu, China) as manufacturer instructions.

Total protein was extracted from lung tissue using RIPA lysis buffer (SPARKjade, EA0002, China). The concentration of the protein samples was detected with the BCA protein assay kit (SPARKjade, EC0001, China). The proteins were then separated by sodium dodecyl sulfate–polyacrylamide gels and transferred to a polyvinylidene fluoride membrane which was blocked with 5% BSA for 1.5 h at room temperature. Then the membrane was incubated overnight at 4 ℃ with the following specific primary antibodies: anti-β actin (proteintech, 20,536-1-AP, 1:2000, USA); anti-α smooth muscle actin (α-SMA) (cst, 14968 s, 1:2000, USA); antifibronectin (FN) (abcam, Ab45688, 1:5000, UK). After washing with TBST, the membrane was incubated with the goat anti-rabbit IgG (H + L) HRP specific secondary antibody (SPARKjade, EF0002, 1:5000, China) for 1 h at room temperature. The Tanon Automatic Image Analysis System (Tanon 5200, China) was used to detect the protein signal and β-actin served as internal reference. The optical density of the protein bands was analyzed using Image J.