Semi dry electrophoretic transfer cell

HCN cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA buffer, Sigma-Aldrich) with 1× protease cocktail inhibitors (Thermo Scientific, Waltham, MA, USA) and 1× phosphatase cocktail inhibitors (Thermo Scientific), 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride for 30 min on ice. After centrifugation (16,100 × g, 10 min), protein concentrations were measured using the BCA protein assay reagent (Thermo Scientific). Proteins were loaded into the gel and electrotransfered to polyvinylidene difluoride (PVDF) membrane with a semi-dry electrophoretic transfer cell (Bio-Rad, Richmond, CA). Membranes were blocked for 1 h at room temperature in a blocking solution consisting of 5 % nonfat dry milk, 0.1 % Tween 20, and Tris-buffered saline (TBST). The membranes were then incubated overnight with the primary antibodies diluted in the blocking solution and washed three times for 10 min. The membranes were incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies diluted in blocking solution. After washing, protein expression was detected by using a chemiluminescence detection kit (Thermo Scientific).

The expression of anti-tumor molecules endostatin and/or TRAIL by engineered Salmonella bacteria was analyzed by western blotting. When freshly cultured bacteria grew to an OD₆₀₀ value of 1.0, 2 ml of bacterial culture was taken. Bacterial pellets were obtained after centrifugation (12,000 × g for 10 min) and proteins present in the supernatant medium were precipitated by trichloroacetic acid (TCA; Sanchez et al., 2009 (link)). Bacterial pellets and concentrated supernatant proteins were boiled in 200 μl loading buffer for 10–15 min, and 10 μl of samples were taken for separation by 12% SDS-PAGE gel. Proteins were then transferred to 0.22 μm nitrocellulose membranes using semi-dry electrophoretic transfer cell (Bio-Rad, China). The membranes were probed with mouse anti-endostatin (MA1-40230, Themofisher) or anti-TRAIL monoclonal antibody (ab2219, Abcam), followed by a horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1030-05, Southern Biotech). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) substrate (BL520A, Biosharp) and visualized by the GelDoc XR+ imaging system (Bio-Rad, China).

Harvested HCN cells were lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich, 89901) containing 1 × protease and phosphatase inhibitor cocktails (78444, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. Every 10 min, the lysates were vortexed. After centrifugation (16,000× g, 20 min), the supernatants were collected. Using a BCA protein assay reagent, the protein concentrations were measured using a BCA protein assay reagent (23225, Thermo Scientific). Typically, 5–10 µg of proteins per well were loaded in an SDS-polyacrylamide gel. After running on an SDS-polyacrylamide gel, proteins were transferred to a polyvinylidene fluoride membrane using a semi-dry electrophoretic transfer cell (BioRad, Hercules, CA, USA) and membranes were blocked for 30 min at room temperature with 5% nonfat dry milk. The primary antibodies were prepared according to the manufacturers’ recommendations and the membranes were incubated with primary antibodies overnight. After washing three times for 10 min each, the membrane was exposed for 1 h with peroxidase-conjugated secondary antibodies diluted in 2.5% blocking solution. The membranes were then processed using a chemiluminescence detection kit (34580, Thermo Scientific). All Western blots were quantified using ImageJ (NIH) software (version 1.54d) and normalized to ACTB.