Pierce ripa lysis and extraction buffer

Samples were lysed
in Pierce RIPA lysis and extraction buffer (Thermo Fisher Scientific,
Waltham, MA) supplemented with 1% Halt protease and phosphatase inhibitor
cocktail (Thermo Fisher Scientific, Waltham, MA) and centrifuged at
14,000g for 20 min at 4 °C to clear the lysate.
Protein concentrations were determined using the Direct Detect Infrared
Spectrometer (EMD Millipore Corp., Burlington, MA), and 10 μg
of each sample was denatured in 6× SDS sample buffer (Boston
Bioproducts, Ashland, MA) for 7 min at 90 °C. Proteins were loaded
into a criterion 4–15% tris-glycine gel (Bio-Rad, Hercules,
CA) and separated by SDS–PAGE at 120 V for 135 min. The gel
was transferred to an IBlot2 nitrocellulose membrane (Invitrogen,
Carlsbad, CA), blocked with TBST blocking buffer (Li-cor Biosciences,
Lincoln, NE) for 1 h, and washed three times with TBST. The membrane
was probed with primary antibodies (1:1000 dilution) in antibody dilution
buffer (1:1 TBST blocking buffer and 1× TBST) overnight at 4
°C. The blot was then washed in triplicate with TBST and incubated
for 1 h with secondary antibody (1:10,000 dilution of IRDye 800 anti-mouse
IgG and IRDye 680 anti-rabbit IgG, Li-cor Biosciences, Lincoln, NE)
in antibody dilution buffer. After a final triplicate wash with TBST,
the blot was visualized using the Odyssey CLx imaging system (Li-cor
Biosciences, Lincoln, NE).

Protein lysates were prepared by using Pierce RIPA lysis and extraction buffer (Thermo Fisher Scientific) containing Halt Protease and Phosphatase inhibitors (1×) on 80%–90% confluent T-75 flasks of each cell line. 250 μL of lysis buffer was added to the cells, and then they were scraped into clean microcentrifuge tubes and placed on ice for 10–15 min. Tubes were then agitated at 4°C for 15 min. Then the tubes were centrifuged for 30 min at 4°C. The supernatant was then transferred to fresh centrifuge tubes, and the protein content was quantified and standardized using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Immunoblotting was carried out using standard protocols with ≥30 μg of total protein mixed with Laemmli sample buffer (Bio-Rad) in each well of the Mini-PROTEAN TGX gels (Bio-Rad). Gels were run at 220–250 V for 30–40 min. Activate LF-PVDF membranes in high-performance liquid chromatography (HPLC)-grade methanol for 5 min. Gels were then transferred to LF-PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Antibodies in 3% w/v BSA in 1× TBST were then used to probe the membranes. The following membranes were purchased from Cell Signaling Technology (β-actin, GAPDH) or Santa Cruz Biotechnology (TLR4, TLR9). Blots were then visualized on a Bio-Rad ChemiDoc Imager.