Ecl detection reagent

Indicated cells were lysed in RIPA lysis buffer (Boster, China) with protease inhibitors. The lysates isolated from secondary generation of mammospheres or tumor tissues were separated by 10% SDS‐PAGE and immunoblotted with appropriate antibodies. The specific primary antibodies used in this study were as follows: SQLE (BS71537, Bioworld, 1:500), KLF4 (11880‐1‐AP, Proteintech, 1:500), c‐Myc (10828‐1‐AP, Proteintech, 1:1000), Sox2 (ab97959, Abcam, 1:500), PCBP2 (ab95942, Abcam, 1:1000), Akt (9272, CST, 1:1000), p‐Akt (12 694, CST, 1:1000), PI3K (4255, CST, 1:1000), p‐PI3K (11 508, Signalway Antibody, 1:500), incubated at 4 °C overnight. The membranes were then incubated with the appropriate horseradish peroxidase‐conjugated secondary antibody for 1.5 h. The proteins were visualized by the ECL detection reagents (Bio‐rad, America) on the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Protein extraction was performed as follows: after treatment with DCMPI (30 μg/mL, 60 μg/mL) for 24 h, HT-29 cells were cultured, collected, rinsed twice with ice-cold PBS, lysed, incubated in RIPA buffer containing a 1% protease inhibitor cocktail for 30 min on ice, and then centrifuged at 12,000 × g for 15 min. The supernatants were harvested and prepared by mixing with 5× sample buffer for subsequent Western blot analysis. The protein samples were then separated by 10% SDS-PAGE and transferred onto 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). The membranes were blocked with 5% non-fat skim milk for 1 h at room temperature and then incubated with the appropriate primary antibodies overnight at 4°C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h, followed by rinsing three times with 1× TBST. The protein bands were visualized utilizing ECL detection reagents (Bio-Rad, United States).

The TH protein expression was determined in CHO cells 30 h after transient transfection with the TH containing plasmids in combination with the proteasome inhibitor Lactacystin (10 μM, Enzo) to inhibit Ub-mediated proteasomal degradation of TH. Cells were lysed (lysis buffer containing 100 mmol/l KCl, 20 mmol/l NaCl, 2 mmol/l MgCl₂, 0.96 mmol/l NaH₂PO₄, 0.84 mmol/l CaCl₂, 1 mmol/l EDTA, 25 mmol/l HEPES, pH 7.2 and protease inhibitor cocktail (SigmaAldrich), 30 sec ultrasound), centrifuged (10,000 g, 4°C, 10 min) and 30 μg total protein was used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE, BioRad). After proteins were transferred onto a nitrocellulose membrane (iBlot, Invitrogen), blocking buffer (5% BSA, 0.1% Tween 20 in 1×TBS) was added for 1 h at room temperature, followed by incubation with anti-TH Ab (polyclonal human and mouse specific, diluted 1:1,000 in blocking buffer; CellSignaling) at 4°C, overnight and washed three times in 1×TTBS (0.1% Tween 20 in 1×TBS). Subsequently, peroxidase-labeled anti-rabbit IgG Ab (CellSignaling) was added (1:2,000 in blocking buffer). The membrane was washed three times (1×TTBS, 5 min, room temperature) and signals were analyzed with the ECL Detection Reagents (BioRad). Blots were analyzed using ImageLab 3.0 software (BioRad). Lysate of murine adrenal gland served as positive control.

The ileum tissues were completely minced with lysis buffer on ice. After the lysate was centrifuged at 4°C and 12,000 g for 10 min, the supernatant was collected. Nuclear protein extraction was performed by the instructions provided with the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGen Biotech, China), and the protein concentration was determined using the BCA method. Extracts containing equal quantities of proteins (25 µg) were electrophoresed in 8 or 12% polyacrylamide gel. Subsequently, the separated proteins were transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk powder in TBS-Tween 20 buffer for 1 h and then incubated with a rabbit anti-IL-1β monoclonal antibody (1:1000 dilution; Proteintech, USA) overnight at 4°C. The membrane was subsequently incubated with secondary antibody (1:5000; Dingguo Biotechnology, China) for 1h at room temperature. Blots were developed using ECL detection reagents (Bio-Rad, USA).

The transfected cells were lyzed in SDS sample buffer, and proteins were separated on a 10% SDS-PAGE and transferred into PVDF membranes. A goat polyclonal antiserum against HSVtk (Santa Cruz Biotechnology, Santa Cruz, CA) and donkey anti-goat IgG-horseradish peroxidase conjugates (Santa Cruz, CA, USA) were used to visualize thymidine kinase. Detection of reactive bands was facilitated by using a horseradish peroxidase-linked secondary conjugate and ECL detection reagents (Biorad, USA).
mGM-CSF and hGM-CSF produced by transfected cells were measured by ELISA of culture medium using a commercial kit (R&D Systems, Minneapolis, MN).

Cells were rinsed with ice-cold PBS buffer thrice and lysed with RIPA lysis buffercontaining 1 mM PMSF on ice for 30 min. The cell debris was subsequently removed by centrifugation at 12000g for 10 min at 4 °C. Then the supernatant was collected for analysis by adding using 5 × SDS loading buffer containing 7% β-mercaptoethanol. Equal amounts of protein samples were separated by 10% (v/v) SDS-PAGE and transferred onto PVDF membranes (0.22 μm, Millipore, USA). After blocking with 5% BSA at room temperature for 2 h, the membranes were incubated with the corresponding primary anti-ZO-1 (1:1000; Proteintech, USA), anti-GAPDH (1:8000; Sigma, USA) and anti-Occludin (1:1000; Proteintech, USA) antibodies at 4 °C overnight. The membranes were then incubated with HRP-conjugated anti-rabbit IgG (1:4000; Cell Signaling Technology, USA) secondary antibodies for 2 h at room temperature, and the immunoreactive protein bands were visualized using ECL detection reagents (Bio-Rad, USA). The intensity of protein bands was quantitated using an Image Lab analysis software and normalized to GAPDH.