Ecl plus reagent

Western blotting was used to detect the presence of UPR pathway proteins in rat livers at each time point, as previously described (10 (link)). Briefly, total protein was extracted using radio immunoprecipitation assay (RIPA) lysis buffer, and the concentration was determined using a bicinchoninic acid (BCA) protein assay kit. Equal amount of proteins were then separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After transfer, the membrane was cut into several strips to detect different target proteins depending on the molecular weight. Hspa41, Hspa1l, c-Jun, and Mapk10 were run together with β-actin; so is Creb3l3 and Creb3l1; Atf2, Atf6, eIF2, and p-eIF2α; xbp1 and Grp78. The membrane was then blocked with 5% bovine serum albumin (BSA) and probed with the primary antibodies (Table 3), followed by probing with horseradish peroxidase-conjugated second antibodies for 2 h at room temperature. The antigen-antibody complexes were visualised using enhanced chemiluminescence (ECL) plus reagent (Millipore, Billerica, MA, United States). Gel-Pro analyser (Bio-Rad) was applied for analysis of the optical density of the protein bands with β-actin as the reference gene. The result was presented as the ratio of the targets to β-actin.

Tissues and whole cell protein were extracted using RIPA lysis buffer, and then cell protein concentration was analyzed by BCA Protein Assay Kit (Beyotime, P0010S). After protein was transferred to PVDF membrane (0.45 or 0.22 mm), the membranes were blocked with 5% nonfat dry milk. Sequentially, the membranes were incubated with primary anti-IL-1β, TNF-α (Santa Cruz, USA), Bcl-2, Bax (OriGene, Herford, Germany), P65, LC3B, P62, p-AKT, AKT, p-mTOR and mTOR (Cell Signaling Technology, USA), β-actin (Beyotime, China) overnight at 4 °C. The membranes were incubated with the respective secondary antibodies for 2 h at room temperature. Finally, the membranes were measured by an ECL plus reagent (Millipore, USA) and the results were detected by the software.

Protein samples were extracted with RIPA-buffer (Heart, China) from LTP-treated cells after 24 h in culture, then heated at 95 °C °C for 5 min with sample buffer (250 mM Tris-HCl, pH 6.8, 10% glycerol, 4% sodium dodecyl sulfate, 2% β-mercaptoethanol, and 0.003% bromophenol blue). Proteins were separated on 12% SDS-PAGE, and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA), and then blocked with 5% dried skim milk in Tris buffer saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. The blocked membranes are rinsed three times in TBST for 10 min, and then incubated in the primary antibodies (anti-phospho p65, 1:5000, Abcam; anti-NF-κB P65 and anti-NF-κBIα Polyclonal Antibody, 1:2000, ABclonal; anti-cyclinD1, 1:200, Santa Cruz; anti-β-actin, 1:2000, CWbio) overnight at 4 °C. After being washed three times with TBST, the secondary antibody horseradish peroxidase (CWbio) was incubated on the membrane for 2 h at room temperature. Then the PVDF membranes were washed three times with TBST and incubated with ECL Plus Reagent (Millipore, USA) and scanned using the chemiluminescence (BioRad, USA). Immunoreactive products were quantified by image-Pro plus 6.0 software to determine the optical density of protein bands.

Samples of cells and EVs were washed and resuspended in RIPA lysis buffer (Beyotime, Shanghai, China) with a protease and phosphatase inhibitor mixture (Millipore). Proteins were separated based on their molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk powder in Tris-buffered saline containing Tween 20 (TBST) for 2 h, and were then incubated at 4 °C overnight with specific primary antibodies (details are listed in Table S1). The membranes were rinsed in TBST three times (10 min each time), incubated with secondary antibodies at room temperature for 2 h and then washed again in TBST (three times, 10 min each time). Protein expression levels were measured with ECL Plus reagent (Millipore) using a Bio-Imaging System (Bio-Rad, USA).

The cell lysates were collected by lysing cells under RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and mixed with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Roche). After removing the cell debris by centrifuging with 15000 rpm for 30 min at 4°C, proteins were quantified by BCA protein assay (Pierce). Next, electrophoresis was performed on 30% acrylamide/bis-acrylamide gradient gel (TOOLS biotechnology), and then transferred to a PVDF membrane (Millipore). After blocking, the following primary antibodies were used: mouse anti-Cntn1 (1:500, LSBio; 1:500, Proteintech, 13843-1-AP), mouse anti-α-Tubulin (1:1000, Proteintech). α-Tubulin was used as an internal control. Primary antibodies were detected through horseradish peroxidase (HRP)-conjugated secondary antibodies listed below: anti-mouse (1:10000, GeneTex), anti-rabbit (1:20000, Sigma-Aldrich). Signals were generated by ECL-Plus reagent (Millipore), and detected under Luminescence/Fluorescence Imaging System LAS-4000 (Fujifilm). Signal quantifications were performed under Image-J based analysis.

CRC and para-CRC tissue samples or cancer cell lines were lysed in RIPA buffer and resolved by SDS-PAGE. Resolved proteins were transferred to polyvinylidene fluoride membranes and incubated with antibodies against TOX (#ab155768, 1:1,000; Abcam, USA), EMT markers (E-cadherin #3195, Snail #3879, and vimentin #5741, 1:1,000, CST, USA; ZEB1 #21544-1-AP, 1:1000 Proteintech, China), and PI3K/AKT/mTOR pathway molecules (p-PI3K #4228, PI3K #4249, p-AKT, AKT, mTOR #2983, p-mTOR #2974, 1:1,000; CST, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Proteins were visualized with ECL Plus reagent (Millipore, Jaffrey, NH, USA) and normalized to β-actin (Proteintech, China, #66009-1, 1:1000). All experiments were replicated three times.

OVCAR3 and SKOV3 cells transfected with shRNA-CREB or negative control were treated with Roflumilast and/or H89, and then the cells were harvested and, according to the manufacturer's instructions, the whole cell protein extracts were resolved on a 10% SDS denatured polyacrylamide gel and were then transferred onto a nitrocellulose membrane, which were blocked in 5% BSA in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated with Anti-GAPDH (#ab8245, Abcam, USA), FtMt (#ab124889, Abcam, USA), CREB (#ab32096, Abcam, USA), p-CREB (#9198, Cell Signaling Technology, USA) antibodies overnight at 4°C. GAPDH was used as the loading control in the Western blotting. After washing with TBST buffer, the blots were then incubated with HRP-conjugated secondary antibody (#BS12478, #BS13278, Bioworld, China) for 1 h at room temperature. After washing with TBST buffer, the blots were visualized using the ECL-Plus reagent (Millipore, Billerica, MA, USA).