Gel pro analyzer

Cells were lysed in ice-cold RIPA buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS) and 0.5% sodium deoxycolate], containing 2 mM Na₃VO₄, 10 mM NaF and a complete set of protease inhibitors (Roche Diagnostics, Mannheim, Germany). The protein concentration was determined using the DC Protein Assay Reagent (Bio-Rad, Milano, Italy). Aliquots of total proteins were resolved with SDS-polyacrilamide gel electrophoresis, transferred onto polyvinylidene fluoride (PVDF) membrane and blotted for 1 h at room temperature (RT) in Tris-buffer saline containing 0.1% Tween-20 and 5% non-fat powdered milk. Membranes were incubated overnight at 4 °C with primary antibodies anti-p-Erk1/2 (1:500), anti p-Akt (1:1000), anti-beta-arrestin1 (1:1000), anti-beta-arrestin2 (1:1000), anti-Shp2 (1:1000) or anti total Erk1/2 (1:1000). Detection was performed using chemiluminescence with horseradish peroxidase-conjugate secondary antibodies (1:1500). Membranes were stripped and re-probed for loading with anti-alfa-tubulin (1:300,000). Densitometric analysis of the immunoblots was performed using the GelPro-Analyzer (Media Cybernetics, Bethesda, MD, USA).

The protein from intestines was extracted according to a previous study [12 (link)]. Protein concentration was determined using a protein quantification kit (Beyotime, Shanghai, China). The protein samples were subjected to SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Inc., Bedford, MA) by wet electroblotting. After blocking with 5% bovine serum albumin (in Tris-buffered saline containing Tween 20) at room temperature for approximately 2 h, samples were subsequently incubated overnight in primary antibodies: Nrf2 and Keap1 (1:2000, Zen Biotechnology, Chengdu, China), Beclin1, ULK1, LC3Ⅱ/Ⅰ, P62, occludin, ZO-1, and Claudin-2, (1:2000, ABclonal, Chengdu, China). The membranes were subjected to washing three times, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for approximately 2 h. β-actin (1:1000; CST) and Lamin B1 (1:2000, Zen Biotechnology) were considered as the control proteins for total and nuclear protein. The immune response bands were measured by enhanced chemi-luminescence. Quantify protein expression was detected by a Gel-Pro Analyzer (Media Cybernetics Bethesda, MD, USA), and analyzed by the ImageJ gel analysis software [96 (link)].

Protein was extracted from IPEC-J2 cells using lysis buffer (Sigma), according to the manufacturer’s instructions. Samples containing equal amounts of protein were run on 10% SDS-polyacrylamide gel and then transferred to a polyvinyldifluoride membrane (Bio-Rad). After blocking with TBST containing 5% nonfat dried milk for 2 h at 37 °C, the membrane was hybridized with the primary antibody of Cell Signaling (TBK1, catalogue no. 3504; IRF3, catalogue no. 4302; p-IRF3, catalogue no. 4947), incubated overnight at 4 °C, and then washed three times with TBST for 10 min each. After washing, the membrane was incubated with secondary antibody for 1 h at 37 °C, then washed three times with TBS/T for 10 min. Clarity western enhanced chemiluminescence (ECL) substrate (Bio-Rad) was used to visualize the signals. The Gel-Pro Analyzer (Media Cybernetics, Inc. Bethesda, MD, USA) was used to quantify protein expression, and the ratio of target proteins’ expression was normalized to β-actin.

The total protein was quantified by BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Samples of 22 μg of protein were applied to a 10% SDS–polyacrylamide gel electrophoresis and the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, Bedford, United States) after running for 45 min at 120 V. After blocking with 5% skim milk, the PVDF membrane was incubated with corresponding primary antibodies including rabbit anti-LC3B (1:500, ET1701-65), anti-SQSTM1 (1:500, R1309-8), anti-Bcl2A1 (1:500, ET1610-20), anti-caspase 3 (1:500, ER 1802-42), anti-PCNA (1:500, R1306-5), anti-AKT (1:500, EM40507), anti-p-AKT (1:500, ET1607-73), anti-FoxO1 (1:500, ET1608-25, HUABIO, Hangzhou, China) and anti-ac-FoxO1 (1:500, A17406, ABclonal, Wuhan, China). Next, the membrane was incubated with the secondary antibodies. Blots were washed three times and visualized using FDbio-Femto ECL Substrate Kit (FD8030, FDbio, Hangzhou, China). For protein quantification, Gel-Pro Analyzer (Media Cybernetics, United States) was used to quantify and analyze images with β-actin as the internal control.

Statistical analyses were performed by using SPSS 21.0 (IBM, USA) and GraphPad Prism version 6.0 software (GraphPad Software, USA). Experimental data are represented as the mean ± SD. Intergroup differences were analysed by one-way analysis of variance (ANOVA) or the independent-samples t-test. Relationships between CASC11 or RAB11FIP2 expression and clinicopathologic parameters were determined by the χ² test. The linear correlation of gene expression was analysed with Spearman’s correlation coefficient. WB images were quantified by a Gel-Pro Analyzer (Media Cybernetics, USA). IHC images were processed with Image-Pro Plus 6.0 (Media Cybernetics, USA). p < 0.05 was considered significant: *, p < 0.05; **, p < 0.01; ***, p < 0.001.