Versadoc 5000 imaging system

Phosphatase inhibitor/buffer mixture II (Sigma-Aldrich, Munich, Germany) and protease inhibitor cocktail (Roche) were used to harvest the cells. They were resolved by sodium dodecyl sulfate–10 % polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore, Schwalbach am Taunus, Germany). The membranes were then incubated with antibodies to activate phosphorylated ERK1 and ERK2 (p-ERK; Cell Signaling Technology, Danvers, MA, USA), total ERK1 and ERK2 (Cell Signaling Technology, Danvers, MA, USA), phosphorylated AKT (p-AKT; Cell Signaling Technology, Danvers, USA), and total AKT (Cell Signaling Technology, Danvers, USA). Antibodies were detected with the appropriate anti-mouse horseradish peroxidase conjugated secondary antibody enhanced by chemiluminescence (Pierce, Life Technologies, Darmstadt, Germany), and images were captured with a Versa Doc 5000 imaging system (Bio-Rad, Munich, Germany).

The MCF7 and SKOv3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The 2 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin-streptomycin (P/S). The medium, FBS and P/S were from Invitrogen (Carlsbad, CA, USA). All cultures were maintained in a humidified incubator at 37°C with an atmosphere containing 5% CO₂.
The expression of ErbB2 in the MCF7 and SKOv3 cells was examined by immunoblot analsyis. Briefly, cells on 35 mm dishes were washed once with phosphate-buffered saline (PBS), and scraped in 1X SDS sample buffer. Cell lysates were subjected to 6% SDS-PAGE, and immunoblotted with rabbit anti-ErbB2 antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. HRP-conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) was used at 1:2,000. Immunoblotting signals were detected using the VersaDoc 5000 Imaging system (Bio-Rad, Hercules, CA, USA) with an enhanced chemiluminescence detection kit (Amersham Biosciences, Pittsburgh, PA, USA).

CD4+ T cells from NCD and HFD mice were cultured with anti-CD3 and anti-CD28 for 15 min and the cells were lysed in RIPA buffer (Thermo Fisher Scientific) for protein extraction. Protein separation was done by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibodies for pAMPK or β-actin (Cell Signaling Technology; Frankfurt, Germany) overnight. Then the membrane was incubated with an HRP-conjugated secondary antibody (Cell Signaling Technology) for 1 h. Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific), VersaDoc 5000 imaging system (Bio-Rad; Hercules, CA, USA), and Image J program were used for detection, visualizing the membranes and quantification, respectively.

Protein abundance was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, as previously described [17 (link)]. Cells were washed with cold PBS and lysed with cell lysis buffer containing protease inhibitors (Boehringer Mannheim, Indianapolis, IN). Equal amounts of proteins were separated on a 10% SDS-PAGE gel under reducing conditions and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were subsequently blotted using antibodies against GRP94, caspase 3, caspase 7, PARP, or GAPDH and horseradish peroxidase-conjugated secondary antibodies, visualized using TOOLS Ultra ECL-HRP Substrate (BIOTOOLS Co., Ltd., Taiwan), and then detected using a VersaDoc 5000 imaging system (Bio-Rad Laboratories).

Cells were disrupted by sonication in 20 mM Tris–HCl, pH7.5, 8 M urea, 0.1% Triton X-100 and Complete Mini protease inhibitor mixture (Roche). After disruption, the samples were centrifuged at 15,000 g for 10 min and the supernatant fraction was used for immunoblotting. The protein content in the supernatant fraction was determined by Bradford assay (Bio-Rad). SDS-PAGE and Immunoblot analyses were performed as previously described [41 (link)]. The primary antibody against S. elongatus DipM (1:1,000), C. paradoxa DipM (1:1,000) and the HA epitope (Roche, 3 F10, 1:1,000) were diluted as indicated. The primary antibodies were detected by horseradish peroxidase–conjugated goat anti-rabbit or anti-rat antibody. The signal was detected with the ECL Prime Western Blotting Detection System (GE Healthcare) and the VersaDoc 5000 imaging system (Bio-Rad).

Mice liver tissue samples were taken in a 2 mL round bottom tube and homogenized using bead mill MM 400 (Retsch, Hann, Germany) containing TNE buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA; pH 8.0) supplemented with phosphatase and protease inhibitor cocktails (Roche, Indianapolis, IN). TNE buffer containing detergents (0.2% SDS, 1% sodium deoxycholate and 1% Triton-X) was added in equal volumes into the homogenates and kept on rocker for 30 min at 4°C. Lysate was centrifuged for 20 min at 15,000 ×g and the supernatant collected. Protein concentration was determined using RC-DC Protein Assay Kit (Bio-Rad, Mississauga, ON). Protein samples containing 30-50 µg proteins were electrophoresed on SDS-PAGE gels and analysed by Western Blot. Primary Ab used are listed in Supplementary Table S2. HRP-conjugated anti-mouse or anti-rabbit secondary antibodies and enhanced chemiluminescence reagents (ECL) were from GE Healthcare Life Sciences (Pittsburg, PA). Images of western blot were captured by the VersaDOC 5000 imaging system (Bio-Rad).