Hybond p 0.45 pvdf

Immunoblot analysis was previously described (59 (link)). Briefly, confluent cells or exosome pellet was lysed in cold lysis buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate) for 10 min. Aliquots of lysates were incubated with PK for 30 min at 37 °C; PK was stopped by addition of proteinase inhibitors (0.5 mm Pefabloc) and directly precipitated with methanol. For samples without PK treatment proteinase inhibitors were added directly and precipitated with methanol. Precipitated proteins were resuspended in TNE buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA). For the gradient, the PK digestion was carried out after the fractionation. The samples were run on 12.5% SDS-PAGE, electroblotted on Amersham Biosciences Hybond P 0.45 PVDF (Amersham Biosciences, 10600023) and analyzed in immunoblot, using the Luminata Western Chemiluminescent HRP Substrates (Millipore, WBLUF0100).

Approximately 20 mg of liver was shortly homogenized in 700 μl lysis buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2.5% SDS, 10 mM beta-glycerolphosphate, 10 mM NaF, 0.1 mM NaOrthovanadate, 1 mM PMSF, and 1x protease inhibitor cocktail) and treated with benzonase (Sigma E1014). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher 23225). A total of 80 μg protein was mixed with laemmli buffer and separated by SDS-PAGE and blotted onto a PVDF membrane (10600023, Amersham Hybond P0.45 PVDF, lot#G9889898). The membrane was probed with primary antibody against GR (sc-1004, Santa Cruz), FOXO1 (sc-11350, Santa Cruz), TFIIB (sc-225, Santa Cruz) or β−Tubulin (05–661 Merck Millipore), and secondary HRP conjugated antibody (DAKO).

E. coli StdOFF and E. coli StdON were grown in LB broth supplemented with carbenicillin (50 μg/ml) and to induce std fimbrial expression anhydrotetracycline (100 ng/ml) at 37°C until an OD₆₀₀ of 0.6 was reached. 10⁸ bacteria were pelleted, resuspended in PBS, mixed with an equal volume of Laemmli buffer supplemented with 10% DTT, and boiled for 10 min. These whole-cell lysates were spun down and supernatants were loaded immediately onto a SDS-PAGE gel (15%). Proteins were transferred to Hybond-P 0.45 PVDF (Amersham) membranes using a Trans-Blot semi-dry transfer cell (Bio-Rad). After blocking with Roti-block (Carl Roth), membranes were incubated first with anti-StdA [38 (link)] serum (Humphries et al., 2003) diluted 1:500 in blocking buffer, and then with a goat anti-rabbit-HRP conjugate, and finally with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Images were obtained using the ImageQuant LAS 4000 system (GE Healthcare).