Skim milk

Protein was extracted from isolated yolk sacs in a lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% Triton X-100) containing Complete Protease Inhibitor cocktail (Roche). After centrifugation at 4°C for 15 min at 20,000×g, the concentration of the supernatants was measured by use of a Micro BCA Protein Assay kit (Pierce). Proteins were denatured in a sample buffer for 5 min at 95°C, resolved by means of SDS-PAGE, and transferred onto an Immobilon-P PVDF membrane (Millipore). The membranes were blocked in 5% skim milk (Nakalai Tesque) in PBS containing 0.1% Tween 20 (Sigma-Aldrich) for 1 h and incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Signals were detected by use of ECL Western Blotting Detection Reagent (Amersham) according to the manufacturer's instructions.

Total protein was extracted from STC‐1 and Lu135 cell lysate. Protein concentration was determined using BCA assay reagents (TAKARA, Tokyo, Japan), and 20 μg protein samples were loaded into 4–12% Bis‐Tris gel (Invitrogen, Carlsbad, CA). After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen). The membrane was blocked by 5% skim milk (Nacalai Tesque, Kyoto, Japan) prior to incubation with rabbit monoclonal antibodies, anti‐PD‐L1‐antibody (clone: E1L3N, Cell Signaling Technology; diluted 1∶1000), and anti‐β‐actin‐antibody (clone: #4967, Cell Signaling Technology; diluted 1:3000), for 1 h at room temperature. Immunoblots were detected using SuperSignal West Femto kit (Thermo Scientific, Rockford, IL).

The 10 μg of cell lysates were subjected to SDS-polyacrylamide gel for electrophoresis by using polyacrylamide gels (5–20%; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The blocking was performed by using 4% skim milk (Nacalai Tesque, Inc.) in PBST. The membranes were incubated with 10 μg/mL of C₄₄Mab-6, 10 μg/mL of C₄₄Mab-46, or 1 μg/mL of an anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corp.) and then incubated with peroxidase-conjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.). Finally, the signals were enhanced by using a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation), and were detected by a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan).

Cells were cultured on coverslips coated with Alcian Blue 8GX (A5268; Sigma-Aldrich), fixed with 4% paraformaldehyde in 1x PBS for 15 min at room temperature, and washed with 1x PBS three times. The fixed cells were permeabilized using 0.2% Triton X-100, 0.02% skim milk (Nacalai), and 0.02% BSA (Sigma-Aldrich) in 1x PBS for 5 min at room temperature in dark. After rinsing with 1x PBS once and then with PBST (0.1% Tween-20, 1x PBS), the cells were incubated with the following primary antibodies diluted in PBST for 45 min at room temperature: rabbit anti-phospho-STING (Ser366) (19781S; 1:200; Cell Signaling Technology); rabbit anti-H3K79me2 (ab2594; 1:200; Abcam); rabbit anti-H3K27me3 (9733T; 1:200; Cell Signaling Technology); and mouse anti-TREX1 (sc-271870; 1:200; Santa Cruz). After three washes with PBST, the cells were incubated with Alexa Fluor 488–conjugated goat anti-rabbit (A11034; Invitrogen), Alexa Fluor 594–conjugated goat anti-rabbit (ab150080; Abcam), or Alexa Fluor 568–conjugated goat anti-mouse (A11031; Invitrogen) at a 1:1,000 dilution in PBST for 45 min at room temperature in dark, and then washed with PBST and Milli-Q water. After air drying, coverslips were mounted on glass slides using PNG anti-fade supplemented with 0.1 μg/ml DAPI. For pSTING-S366, the average intensity within a cell boundary was analyzed by ImageJ software.

The cell lysates (10 μg of protein) were prepared using sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.), and separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were incubated with 10 μg/mL of C₄₄Mab-108, 1 µg/mL of an anti-PA16 tag mAb (NZ-1), or 1 μg/mL of anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corp.) in 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20. Then, the membranes were incubated with anti-mouse immunoglobulins conjugated with peroxidase (diluted 1:1000; Agilent Technologies, Inc.) for C₄₄Mab-108 and anti-β-actin. The anti-rat immunoglobulins (diluted 1:10,000; Sigma-Aldrich Corp.) conjugated with peroxidase were used for NZ-1. Finally, the signals were detected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Protein was isolated on ice using cold RIPA buffer (#R0278, Sigma-Aldrich, St Louis, MO, USA) supplemented with protease (#539134) and phosphatase (#524625) inhibitor cocktails (Calbiochem, San Diego, CA, USA). Protein concentration was quantitated using the BCA Protein Assay Kit (#23225, Thermo Fischer Scientific). SDS-PAGE electrophoresis was performed using 7.5% acrylamide gels that were transferred onto PVDF membranes (#IPFL00010; Millipore, Billerica, MA, USA). Immunoblotting was performed by blocking the membranes with 5% skim milk (Nacalai Tesque) diluted in Tris-buffered saline (TBS), followed by incubating with mouse monoclonal anti-E-cadherin (#610182, BD Biosciences, Franklin Lakes, NJ, USA; 1:2500) or mouse monoclonal anti-β-actin (#A1978, Sigma-Aldrich; 1:5000). Following washing steps, the membranes were then incubated with IRDye 800CW-conjugated (#926-32210) or IRDye 680-conjugated (#926-32220; LI-COR Biosciences, Lincoln, NE, USA) goat anti-mouse antibodies. Following final washing steps, the western blots were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences).