Non fat dried milk

Anti-SOCS1 (3950S, CST, Danvers, MA, United States), anti-SOCS3 (52113, CST, Danvers, MA, United States), anti-p-Stat1 (9167, CST, Danvers, MA, United States), anti-Stat1 (9172, CST, Danvers, MA, United States) and anti-β-actin (3700S, CST, Danvers, MA, United States) were used in this study. The secondary antibodies are HRP-labeled anti-rabbit and anti-mouse antibodies (CST, Danvers, MA, United States). The total proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Roche, Germany). The PVDF membrane was blocked with TBST (2.42 g/L Tris base, 8 g/L NaCl, 0.1% Tween 20, pH 7.6) containing 5% non-fat dried milk (BD, Franklin Lakes, NJ, United States) at room temperature for 2 h. After washing three times with TBST, the membrane was incubated overnight with primary antibodies at 4°C, and incubated with the secondary antibody for 1 h at room temperature. After washing with TBST, the membrane was developed with an enhanced chemiluminescence kit (TIANGEN, Beijing China) in a dark room. The intensity of signaling was quantified by ImageJ 1.8.0 software (National Institutes of Health, United States).

HeLa cells stably transfected with the indicated plasmids were lysed using RIPA lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 supplemented with 1 × cOmplete protease inhibitor cocktail (Sigma-Aldrich). The cell lysates were electrophoresed via SDS–PAGE, transferred onto PVDF membranes (Amersham), blocked with 5% non-fat dried milk (BD Biosciences), and incubated with appropriate primary and HRP-conjugated secondary antibodies (Bio-Rad Laboratories). Immunoblots were visualized using an ECL detection system (Santa Cruz Biotechnology). The Western blots were imaged using ChemiDoc MP (Bio-Rad). The following primary antibodies were used: rabbit anti-Myc (A14; Santa Cruz, 1:1,000), rabbit anti-E-cadherin (24E10; Cell Signaling Technology, 1:1,000), anti-vimentin (D21H3; Cell Signaling, 1:1,000), anti-N-cadherin (#32; BD Biosciences, 1:1,000), anti-β-catenin (6B3; Cell Signaling, 1:1,000), anti-β-tubulin (9F3; Cell Signaling, 1:1,000), and anti-DSG1 (H-290; Santa Cruz, 1:1,000). The full-length blots are shown in Supplementary data, Fig. S13.

Human THP-1 cells (5×10⁶ cells) were cultured in 10 cm dish and harvested after R848 stimulation as indicated time points and lysed in 1 ml RIPA buffer (50 mmol/l Tris-HCl pH7.4, 150 mmol/l NaCl, 1% sodium deoxycholate, 0.5 mol/l EDTA, 1 mmol/l NaF, 1% Nonidet P-40, supplemented by 10% proteinase inhibitors cocktail). 1/10 lysate (100 μl) was reserved for direct immunoblot analysis while the rest was successively incubated with rabbit anti-TLR7 or normal rabbit IgG antibodies for 4 h and with protein G agarose (GE Healthcare) for another 1 h. After 5 rounds of washes, proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membrane (Amersham, Piscataway, NY). Nonspecific sites were blocked with 5% nonfat dried milk (BD Biosciences, San Jose, CA) for 1 h at room temperature. After 3 times of PBS wash, the nitrocellulose membrane was incubated with primary and secondary antibodies. Blots were analyzed using a Luminescent Image Analyzer (Fujifilm LAS-4000).

Radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors was used in order to lyse and extract protein from murine brain homogenates and cultured cells. Supernatant protein concentrations were then determined using a BCA assay kit, and protein lysates (100 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose (NC) membranes (Amersham, Piscataway, NY, USA). Nonspecific binding was blocked with 5% nonfat dried milk (BD Biosciences, San Jose, CA, USA) for 1 h at room temperature. After three washes in PBS, the NC membranes were incubated with appropriate primary and secondary antibodies. Blots were then analyzed using a Luminescent Image Analyzer (Fujifilm LAS-4000; GE Life Sciences, Piscataway, NJ, USA). The relative target protein expression to internal control is quantified using Image J software.