Nitrocellulose nc membrane

To detect the role of PA-MSHA in murine bone marrow (BM)-derived macrophages, macrophages were separated and stimulated with different concentrations of PA-MSHA in vitro. To further explore whether PA-MSHA stimulation efficiency is time-dependent, murine BM-derived macrophages were isolated and cultured with 10⁶/mL PA-MSHA for 0, 6, 12, 24, 48, and 72 h. Then total RNA were extracted using TRIzol reagent (Invitrogen, USA), and cDNA were synthesized using one step PrimeScript miRNA cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). The expression levels of cytokine (IL-12, TNF-α, IFN-γ, IL-4, IL-10 and TGF-β) were measured by qRT-PCR and the dates were determined by the 2^−ΔΔCT method [26 (link)]. All qRT-PCR reactions were performed three times. Subsequently, the protein expression levels of the cytokines were determined by Western blotting. In brief, cells in culture were lysed in RIPA buffer (Sigma, USA), then, the proteins were loaded onto 10% SDS-PAGE prior to being transferred onto nitrocellulose (NC) membranes (Amersham Bioscience, UK). Following blocking with skimmed milk, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. GAPDH was used as a control. Each protein sample was examined in triplicate.

Western blot analysis was performed to evaluate the expression of genes at protein level. Total protein was extracted from cells or murine tumor tissues by using RIPA (Radio Immunoprecipitation Assay) lysis buffer with protease inhibitors, followed by the concentration determined with bicinchoninic acid (BCA; Beyotime, China) method. Thereafter, the separation of proteins was realized by 10% SDS-PAGE, with the separated proteins transferred to nitrocellulose (NC) membranes (Amersham Bioscience, UK) subsequently. Following being blocked by 5% skim milk, the membranes were incubated at 4 °C overnight with primary antibodies specifically against E-cadherin, N-cadherin, Vimentin, EZH2 and GAPDH (All from Abcam, Cambridge, UK). At length, the visualization of chemiluminescent signals was performed using ECL (Enhanced Chemiluminescent) detection reagents (Amersham Biosciences, Sweden). GAPDH acted as the internal control. Proteins in each sample were detected in triplicate.

Whole cell protein extraction and western blotting were performed as previously described [44 (link)]. BV-2 cells were seeded at 5 × 10⁶ cells/mL in 6-well plates for 24 h. The cells were pre-treated with ZB5-1 (10, 50, and 100 μM) for 1 h and then activated by LPS (1 μg/mL) for another 24 h. The collected cells were rinsed three times with cold phosphate buffer saline (PBS), and total cell lysates were obtained by adding 100 μL of RIPA buffer. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (20–40 µg) were separated electrophoretically using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred onto nitrocellulose (NC) membranes (Amersham). Membranes were then blocked with 5% skim milk and incubated with the primary antibody and the horseradish peroxidase (HRP)-conjugated secondary antibody. Blots were quantified by an Amersham ECL Western Blotting Detection Kit (GE Healthcare).