Sm0671

For each SDS PAGE, prestained standard protein ladder (catalog number SM1811, a mixture of nine recombinant proteins with MWs of 11, 17, 28, 36, 55, 72, 95, 130 and 250 kDa, or SM0671, a mixture of ten recombinant proteins with MWs of 10, 15, 25, 35, 40, 55, 70, 100, 130 and 170 kDa, Fermentas) was loaded along with different protein samples. After gel electrophoresis, an R_f value (the migration distance of a protein divided by the migration distance of the front-running dye) was obtained for each standard control protein. The R_f value was plotted against the lg(MW) of corresponding standard control protein to get the linear formula lg(MW) = aR_f + b (where a is the slope and b is the y-intercept). The R_f value for each protein sample was then obtained and used for the calculation of the SDS PAGE-displayed MW of the corresponding protein.

After amplification using primers PmrAF and PmrAR, PCR products were inserted into pET-28a(+) digested with NdeI and HindIII to add a carboxyl terminal 6-his-tag. Plasmids were transformed into the expression strain E. coli BL21 (DE3) and cultured in LB medium (5 ml) at 37°C overnight. Cells were diluted 1:100 into 1 L LB broth containing ampicillin and cultured. When OD₆₀₀ was between 0.4 and 0.5, recombinant PmrA was activated with 0.1 mM IPTG at 16°C for 20 h.
Mixtures were centrifuged at 7,000 × g for 20 min at 4°C and resuspended in lysis solution, followed by harvest of 6 L of cells and disruption by a One Shot Cell Disrupter (Constant Systems, Daventry, UK). To isolate insoluble substances, mixtures were passed through 0.45-μm syringe-end filters after centrifuging at 12,000 × g for 20 min at 4°C before loading on a His-tag column. Supernatants were transferred to a Ni-NTA affinity chromatography column (GE Healthcare, Pittsburgh, PA, USA). After balancing the column with lysis buffer, recombinant protein was added and eluted using washing buffer with an imidazole concentration gradient (0, 30, 60, 100, 200, and 500 mM). Washing buffer (30 ml) containing target recombinant proteins was dialyzed in 20 mM Tris-HCl, pH 8.0. Proteins were subjected to SDS-PAGE. Unstained and prestained protein markers (Fermentas, SM0431 and SM0671 Waltham, USA) were applied as references.

Whole-cell lysates were prepared for extraction by incubation in RIPA lysis buffer for 30 min at 4 °C with protease inhibitor cocktail (P8340, Sigma) and phosphatase inhibitor cocktail (Sigma, P5726). Proteins were separated by a 4–12% Novex Bis-Tris pre-cast polyacrylamide gradient gel (Life Technologies) and transferred to a polyvinylidene difluoride membrane using iBlot dry blotting system (Life Technologies). Molecular weight was indicated by using a pre-stained protein ladder (SM0671, Fermentas). After blocking 1 h in room temperature, the membrane was incubated with primary antibodies recognizing Smad2/3 (1:1,000, #3102, Cell Signaling), pSmad 2 (1:1,000, #3101, Cell Signaling), β-actin (1:5,000, A-5441, Sigma) or ER (1:500, NCL-ER-6F11/2, Novocastra) in blocking solution with gentle rocking overnight at 4 °C. Secondary antibodies conjugated with horseradish peroxidase (Dako) were incubated for 1 h at room temperature. Western blots were visualized using enhanced chemiluminescence solution (PerceECL 32106, Thermo Scientific) and a chemiluminescence imager (Amersham Image 600, GE Healthcare life sciences).