Tris glycine gel

For protein quantification, we proceeded as described before^{62 (link)}. Indeed, protein concentrations were determined with a standard Bradford assay and samples were boiled for 10 min in Laemmli buffer. A total of 15 μg of samples were loaded on 4–15% Tris–glycine gels (Bio-Rad), run 1 h at 100 V, transferred onto nitrocellulose membranes and blocked for 1 h in Odyssey blocking buffer diluted 1:1 in Tris buffer saline (TBS). After blocking, membranes were incubated with primary antibodies diluted in blocking buffer, overnight at 4 °C [rabbit polyclonal anti-Mortalin (Origene Technologies, 1/5,000); mouse monoclonal anti–β-actin, (Sigma–Aldrich, 1/100,000); mouse monoclonal anti-total ERK1/2 (Cell Signaling, 1/2,000) and rabbit anti-ERK1/2 phThr202/Tyr204 (Cell Signaling, 1/2,000); mouse monoclonal anti-Hsp60 antibody (Santa Cruz Biotechnology, 1/1000)]. Membranes were then washed extensively in Tween-20 containing TBS (TBST) and incubated for 1 h at RT with secondary antibodies: goat anti-rabbit Alexa 680 and goat anti-mouse Alexa 770 (Biotium, 1/15,000) both diluted in Odyssey blocking buffer (1:1 v/v TBS). Laser scanning and analyses of the blots were performed using the Odyssey Infrared Imaging System (Li-Cor).

Enriched splenic DCs were cultured in 96- or 24-well plates with purified flagellin (1 μg/mL or 10 ng/mL) and lysed at various time points with RIPA buffer (Sigma) containing protease inhibitors (Sigma). Whole-cell extracts were centrifuged, loaded onto 4–15% Tris/glycine gels (Bio-Rad, USA), and transferred to polyvinylidene fluoride or nitrocellulose membranes (PVDF)(GE Healthcare, Pittsburgh, PA, and Invitrogen). All antibodies (anti-phospho PKC-delta, anti-caspase-1, anti-β-actin, anti–TEK, and anti-phospho-IRS-1) were added at 1:1000 dilution, followed by HRP secondary antibodies (Jackson Immuno Research, West Grove, PA). ECL plus (GE Healthcare) was used as substrate and blots examined using a Kodak Image Station.

Protein extracts were prepared as previously described [10 (link)]. For each lane, 20 μg of total cell lysates were separated in 4–15% Tris–glycine gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 100 V. Proteins were then transferred to 0.2 μm PVDF membranes (Bio-Rad Laboratories, Inc.), probed with the specific primary antibodies, followed by secondary antibodies conjugated with horseradish peroxidase according to manufacturer’s indications. Primary antibodies used for Western blot include MDM2 (Santa Cruz Biotechnology, #sc-965, Santa Cruz, CA, USA), Phospho-c-Jun (Ser243) (Cell Signaling, #2994, Danvers, MA, USA), p53 (Cell Signaling, #2524), p21^Waf1/Cip1 (Cell Signaling, #2947), p27^Kip1 (BD Biosciences, #K25020). β-Actin (Cell Signaling, #3700) was used as the loading control. All the antibodies were used at working concentration indicated by manufacturers. Protein bands were detected by Clarity western ECL (Bio-Rad Laboratories, Inc.) and the blots were semi-quantified with ImageJ software. All the experiments were performed in biological triplicate.

Frozen cell lysates were boiled at 95°C for 10 min followed by centrifugation at 15,000 rpm for 10 min to clear cellular debris. The resulting supernatant was run on precast 4-15% Tris Glycine gels (Biorad) at 200V for 1h. Proteins were transferred to nitrocellulose membranes at 100V for 1h. Following transfer, the blots were blocked with Tris-buffered saline with 0.1% Tween-20 detergent (TBST) + 5% milk and probed overnight at 4°C using TBST + 5% milk with anti-MgtA (1:2,500 ; generated in this study). After overnight incubation, blots were washed 3 times with TBST for 5 min and probed with goat anti-rabbit HRP antibody (1:10,000) for 1 h at room temperature. Blots were washed 3 times with TBST for 5 min and visualized using a ChemiDoc MP Imaging System (Biorad).