Erk1 2

(1) After the cells in each group were washed with PBS for 3 times, RIPA lysate (Meilunbio, Dalian, China) was used to isolate total protein from HA-VSMCs, and extracted proteins were quantified by a BCA Protein Quantification Kit (Epizyme, Shanghai, China). (2) SDS-PAGE gel (Epizyme, Shanghai, China) electrophoresis was performed, and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% slim milk for 1 h. (3) The membranes were incubated overnight at 4 °C together with relevant primary antibodies and then incubated with corresponding secondary antibody for 2 h at room temperature. (4) The protein bands were visualized by enhanced chemiluminescence (Vazyme, Nanjing, China) and quantified by Image Lab (LI-COR). β-actin, PCNA and MMP-9 antibodies were purchased from Absin (Beijing, China), while Notch1, Jagged-1, ERK1/2 and p-ERK1/2 were from Abmart (Shanghai, China).

To investigate the rTsTryp degrading or down-regulating expression of gut epithelium TJs proteins and related pathways, soluble proteins from Caco-2 monolayers pretreated with 20 μg/ml rTsTryp at 37°C for 2 h were analyzed by western blotting as described before [14 (link),37 (link)]. Briefly, Caco-2 cell monolayers were incubated with rTsTryp at 37°C for 2 h, cell proteins were collected, and the cell lysates were separated by 10% SDS-PAGE and transferred subsequently onto PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk in TBST for 1 h at 37°C, and cut into strips. Subsequently, the strips were probed overnight at 4°C with antibodies against ZO-1 (1:1 000, Servicebio, Wuhan, China), E-cad (1:200, Santa Cruz, USA), occludin (2 μg/ml, Santa Cruz), claudin-1 (2 μg/ml, Santa Cruz), PAR2 (1:1 000, Abcam, UK), p-ERK1/2 (1:1 000, Abmart, Shanghai), ERK1/2 (1:1 000, Abmart, Shanghai), GAPDH (1:1 000), and β-actin (1:2 000) (Servicebio). After washing with TBST, the strips were incubated with HRP -conjugated anti-mouse IgG or anti-rabbit IgG as secondary antibodies (1:10 000). Finally, the color of the strips was developed using an enhanced chemiluminescence kit (CWBIO, Beijing, China). The results for the above proteins were normalized using GAPDH as a loading control [4 (link),18 (link)].

Total protein was extracted from cell samples using RIPA lysis buffer (Beyotime Biotechnology) supplemented with Thermo Scientific Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Protein extracts were resolved by SDS-PAGE and then electrophoretically transferred onto a PVDF membrane (EMD Millipore). The membrane was incubated for 2 h in blocking buffer (1 × TBST containing 5% non-fat milk), and then was incubated at 4℃ overnight with following primary antibodies: MIAC (abmart, 1:1000), AQP2 (abcam, ab199975), EREG (Cell Signaling, 12,048), EGFR (Proteintech, 18,986–1-AP), Phospho-EGFR (Abmart, T55232), mTOR(Abmart, T55306), p-mTOR (Abmart, T56571), Akt (Abmart, T55561), Phospho-Akt (Abmart, T40067), ERK1/2 (Abmart, T40071), Phospho-ERK1/2 (Abmart, TP56192), GAPDH (Proteintech, 60,004–1-Ig), HA (Abmart, M20003S). The immunocomplexes were subsequently incubated with the HRP-conjugated secondary antibodies, and detected with the Western Blot Detection kit (Beyotime Biotechnology) and TANON-5200 system (Tanon, Shanghai, P.R. China). The densitometric ratio of protein bands was calculated by ImageJ program.