Mini nitrocellulose transfer packs

Uncropped scans of important gels are shown in Supplementary Fig. 9. Approximately 60 μl pre-competent cultures grown at 37 °C in C+Y medium to OD₅₅₀ 0.1 were incubated with 25 ng ml⁻¹ synthetic competence stimulating peptide (CSP) for 7 min at 37 °C to induce competence. Cultures were centrifuged 5 min at 5,000 g 4 °C, supernatants were discarded and pellets were stored 12 h at −80 °C. Pellets were resuspended with 50 μl buffer (TE 1X and 0.01 Doc) and heated 5 min at 80 °C with loading buffer. 10 μl extracts were then loaded on SDS-PAGE precast 4–20% gels (Mini-PROTEAN TGX gel, Bio-Rad) followed by electrophoresis at 180 V for 35 min in 1X TGS buffer. After migration, proteins were transferred to a nitrocellulose membrane (Mini Nitrocellulose Transfer Packs, Bio-Rad) with the trans-Blot Turbo Transfer System (Bio-Rad). The nitrocellulose membrane was then cut above the 17 kDa marker band. The top part of the membrane was probed with anti-RadA antibodies (1:1,000) and the bottom part with anti-SsbB (1:5,000). Both primary antibodies were raised in rabbit (Eurogentec) from purified protein.

Dulbecco’s modified Eagle’s medium (DMEM) was bought from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). Stable l-glutamine, 1% non-essential amino acids, penicillin/streptomycin, and chemiluminescent reagent were from Euroclone (Milan, Italy). Polycarbonate filters, 12 mm diameter, 0.4 µm pore diameter were from Transwell Corning Inc. (Lowell, MA, USA). Phenol red, PBS, bovine serum albumin (BSA), RIPA buffer, and the antibody against β-actin were from Sigma-Aldrich (St. Louis, MO, USA). The antibody against HMGCoAR was bought from Abcam (Cambridge, UK), that against PCSK9 from GeneTex (Irvine, CA, USA), that against phospho-HMGCoAR (Ser872) from Bioss Antibodies (Woburn, MA, USA), and that against LDLR from Pierce (Rockford, IL, USA). The antibodies against SREBP-2, rabbit Ig-HRP, mouse Ig-HRP, phenylmethanesulfonyl fluoride (PMSF), Na-orthovanadate inhibitors were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); the inhibitor cocktail Complete Midi from Roche (Basel, Switzerland). Mini protean TGX pre-cast gel 7.5% and Mini nitrocellulose Transfer Packs were purchased from BioRad (Hercules, CA, USA). The human proprotein convertase 9 immunoassay (Quantikine ELISA) was bought from R & D System (Minneapolis, MN, USA).

Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin/streptomycin, chemiluminescent reagent and 24- or 96-well plates were purchased from Euroclone (Milan, Italy). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DPPH (1,1-diphenyl-2-picrylhydrazyl), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), TPTZ, Griess reagent, bovine serum albumin (BSA), RIPA buffer, the antibody against β-actin, the fluorometric intracellular ROS kit and MDA assay kit were bought from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethanesulfonylfluoride (PMSF), Na-orthovanadate inhibitors and the antibodies against rabbit Ig-horseradish peroxidase (HRP) and mouse Ig-HRP were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The iNOS primary antibody came from Cell Signaling Technology (Danvers, MA, USA); the inhibitor cocktail Complete Midi from Roche (Basel, Swiss); mini-protean TGX pre-cast gel 7.5% and mini-nitrocellulose Transfer Packs from Bio-Rad (Hercules, CA, USA). Ultrapure water was produced using a Milli-Q System (Millipore SA, Molsheim, France). Acetonitrile with a HPLC grade was from Panreac (Barcelona, Spain); formic acid was from Merck (Darmstadt, Germany). Standards of quercitrin (>98.5%) and ursolic acid (>98.0%) were from Extrasynthese (Genay, France).