Phosphatase inhibitors cocktail

Hep3B cells were seeded in 60-mm plates 5 × 10⁵ cells/plate and treated with apigetrin (0, 50, and 100 µM) for 48 h. After incubation, cells were lysed using RIPA (iNtRON Biotechnology, Seoul, South Korea) containing a protease inhibitor and a phosphatase inhibitors cocktail (Thermo Scientific, Rockford, IL, USA) and centrifuged at 10,000 rpm for 10 min at 4 °C. A PierceTM BCA (Thermo Fisher Scientific, Rockford, IL, USA) was used to determine the concentration of extracted protein. Proteins were separated with SDS polyacrylamide gel (8–15%) electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, 0.45 mm; Millipore, Billerica, MA, USA) utilizing a semi-dry transfer technique (Atto Corp., Tokyo, Japan). These membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 1% Tween 20 (TBS-T, pH 7.4), 5% BSA (bovine serum albumin), or 1X phosphoblocking solution at RT for 1 h and then incubated overnight with primary antibody 1:1000 at 4 °C, then followed by 2 h of secondary antibody at RT. An electrochemiluminescence (ECL) detection device (Bio-Rad Laboratory, Hercules, CA, USA) was used to detect proteins. ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) was used to quantify protein expression.

To detect the post-translational modification of YB-1 protein, two-dimensional (2D) WB was conducted as previously described [45 (link)]. Whole cell lysate of iDC and 27DC were prepared using RIPA (Boston BioProducts, Milford, MA, USA) supplemented with 5 mM EDTA (Quality Biological), 1X protease inhibitors (MilliporeSigma), and 1X phosphatase inhibitors cocktail (Thermo Fisher Scientific) at 10x10⁶ cells/mL for 15 minutes on ice. The cell lysate was centrifuged at 15,000 x g for 10 minutes at 4°C to remove cell debris. Protein concentration was determined using a BCA Assay. The total cellular protein (100 μg) was subjected for 2D-WB analysis [45 (link)].

To detect the post-translational modification of YB-1 protein, two-dimensional (2D) WB was conducted as previously described [45 (link)]. Whole cell lysate of iDC and 27DC were prepared using RIPA (Boston BioProducts, Milford, MA, USA) supplemented with 5 mM EDTA (Quality Biological), 1X protease inhibitors (MilliporeSigma), and 1X phosphatase inhibitors cocktail (Thermo Fisher Scientific) at 10x10⁶ cells/mL for 15 minutes on ice. The cell lysate was centrifuged at 15,000 x g for 10 minutes at 4°C to remove cell debris. Protein concentration was determined using a BCA Assay. The total cellular protein (100 μg) was subjected for 2D-WB analysis [45 (link)].

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).