Las 4000 imager

Cells were harvested in RIPA lysis buffer, and protein was quantified with the BCA assay (Beyotime). Whole-cell lysates containing 15 μg total protein were loaded onto 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels, separated by electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding on the membranes was blocked with 5% bovine serum albumin (BSA) in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Membranes were then probed with primary antibodies (1 : 1000) against mouse FoxO1, DYKDDDDK Tag (Cell Signaling Technology, Danvers, MA, USA) and α-tubulin (Sigma) in blocking solution overnight at 4 °C. After washing with TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Protein bands were visualized by exposing to an enhanced chemiluminescence detection system (LAS-4000 imager, Fujifilm, Tokyo, Japan) using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). Densitometry analyses were performed using ImageJ 1.42q software (National Institutes of Health), and the values for target proteins were normalized to α-tubulin as the endogenous control.

Tissues or cells were lysed with ice-cold RIPA buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxysodium cholate, 0.1% SDS, 5 mM EDTA, 10 mM NaF. Before use, add 1 mM PMSF, 3 mM dithiothreitol, 1 mM sodium vanadate, and protease inhibitors (Merck)]. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes or polyvinylidene fluoride membranes. Immunoblots were developed in chemiluminescence reagent (PerkinElmer Life Sciences) and exposed in a Fujifilm LAS 4000 imager.

Membrane proteins were prepared from LβT2 cells as previously described (Garrel et al., 2016 (link)). Equal amounts of protein (20 μg) were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. Specific antibodies and Pierce ECL2 substrate were used to detect Phospho-CREB (P-CREB; Cell signaling #9198; 1:1000), Phospho-Extracellular signal-regulated kinase1/2 (P-ERK 1/2; Cell signaling #9101; 1:1000), Phospho-p38 (P-p38; Cell signaling #4511; 1:1000), Phospho-Jun-kinase (P-JNK; Cell signaling #4671; 1:1000), Phospho-Smad2 (P-Smad2; Cell signaling #3108; 1:1000). Respective non-phosphorylated proteins, including Total CREB (Cell signaling #9197), Total ERK1/2 (Cell signaling #9102), Total p38 (Cell signaling #8690), Total JNK (Cell signaling #4671), Total Smad2 (Cell signaling #5339) were also detected (dilution of antibodies, 1:1000) as well as vinculin (Sigma-Aldrich #V9131; 1:20 000), used as an internal loading control. Blots were analyzed with a Fuji LAS-4000 imager and quantified using MultiGauje software. Full scans of the entire original gels are presented in Supplementary Materials 1, 2.

Immunoblotting was performed as described previously (35 ). For sample preparation, M. xanthus cells were harvested from exponentially growing suspension cultures and resuspended in SDS lysis buffer. Proteins were loaded from an equal number of cells per sample. As primary antibodies, rabbit polyclonal anti-FLAG (1:2,000; Rockland) and anti-PilC (1:2,000) (38 (link)) antibodies were used, with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (1:15,000, Sigma) as the secondary antibody. Immunoblots were developed using Immobilon Forte Western HRP substrate (Millipore) on a LAS-4000 imager (Fujifilm).

SK-Hep-1 human hepatoma cells were exposed to various concentrations of OPD for the indicated times. After incubation, the cells were lysed, and the protein concentrations were determined by the bicinchoninic acid method [37 (link)]. Each protein was subjected to 6–15% SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA) by electroblotting, and membranes were blocked for 1 h with blocking buffer [5% bovine serum albumin (BSA) in tris-buffered saline-0.1% Tween 20 (TBST)] at room temperature [38 (link)]. Membranes were then incubated with indicated antibodies (mouse anti-β-actin, diluted 1:10,000; other antibodies, diluted 1:500–1:1000 in 5% BSA/TBST) overnight at 4 °C and washed three times for 10 min with TBST. After washing, membranes were incubated with corresponding secondary antibodies diluted 1:2000 in TBST for 2 h at room temperature, washed three times for 10 min with TBST, and visualized with an enhanced chemiluminescence (ECL) detection kit (LabFrontier, Suwon, Korea) using an LAS-4000 Imager (Fuji Film Corp., Tokyo, Japan).