Chemiluminescent substrate

Flow cytometry sorted cells were washed in PBS and resuspended in RIPA buffer, 1 mM PMSF, 1 mM Na₃VO₄, and 1 × protease inhibitor cocktail for 3 min on ice. The lysate was centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was used for western blotting. Protein lysates were boiled in loading buffer (Beyotime, Jiangsu, China), resolved by electrophoresis on 8% SDS-polyacrylamide gels, and transferred to PVDF membranes (Amersham Pharmacia Biotech, Amersham, UK). Membranes were probed overnight at 4 °C with primary antibodies recognizing ERK1/2, phospho-ERK (Thr202/Tyr204), AMPKα, phospho-AMPKα (Thr172), P70S6K, and phospho-P70S6K (Thr389) (Cell Signaling, Danvers, MA, USA), with GAPDH (Cell Signaling) as the control. Horseradish peroxidase-conjugated IgG (Beyotime) was used to detect specific proteins. Finally, immunodetection was conducted using chemiluminescent substrates (Amersham Pharmacia Biotech).

The cells were harvested, washed in PBS, and solubilized in lysis buffer in the presence of phosphatase and protease inhibitors (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% Igepal CA-630, 0.5% Na-Doc, 0.1% SDS, 1 mM Na₃VO₄, 1 mM NaF, 2.5 mM EDTA, 1 mM PMSF, and 1× protease inhibitor cocktail). After incubation on ice for 30 min, the lysates were centrifuged at 14,000× g for 10 min at 4 °C and the supernatant fractions were used for Western immunoblot analysis. The protein extracts (30 μg/lane) were resolved on a 12% SDS-polyacrylamide gel and electro-blotted onto PVDF membranes (Millipore, Milan, Italy). The membranes were blocked in 5% low-fat milk in TBST (50 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20) for 1 h at room temperature and probed overnight at 4 °C with rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000) (Cell Signaling, Danvers, MA, USA, #5174S) and mouse monoclonal anti-vinculin (Santa Cruz Biotechnology, Heidelberg, Germany, sc-66305) antibodies. Horseradish peroxidase conjugated anti-rabbit or anti-mouse IgGs (1:3000 in blocking solution) (Upstate Biotechnology, Milan, Italy) were used as a secondary antibody. Immunodetection was carried out using chemiluminescent substrates (Amersham Pharmacia Biotech, Milan, Italy) and recorded using a HyperfilmECL (Amersham Pharmacia Biotech).

The cpxR gene was cloned into the pET28a(+) plasmid (Novagen, Madison, WI, USA), and the recombinant proteins were expressed in E. coli BL21 (DE3) cells by addition of 1 mM isopropyl-β-d-thiogalactopyranoside. The purification of CpxR fusion protein was performed with a HisTrap high-performance column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) as previously described [14 (link)]. The CpxR protein was phosphorylated with acetyl phosphate (Sigma, St. Louis, MO, USA) as previously described [51 (link)]. Then, EMSA were performed to determine the binding of phosphorylated CpxR (CpxR-P) to the hcp2B promoter. Briefly, the sequence of the hcp2B promoter region with or without the putative CpxR binding site was amplified and labeled with biotin. The biotin-labeled DNA probe (40 ng) was incubated with increasing concentrations of CpxR-P protein in EMSA binding buffer (10 mM Tris, 50 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM MnCl₂, 2.5% glycerol and 50 ng/μL poly[dI-dC]). After incubation for 30 min at room temperature, the reactions were subjected to electrophoresis and transferred to a nylon membrane. The biotin-labeled DNA was detected with a chemiluminescent substrate (Amersham Pharmacia Biotech). A competitive EMSA was performed by simultaneously incubating the biotin-labeled and unlabeled hcp2B promoter region with CpxR-P protein.