Ecl substrate

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor (Cat. 25765800, Sigma Aldrich) and a phosphatase inhibitor (Cat. P5726, Sigma Aldrich). After denaturing the samples at 95°C for 5 minutes, 30μg of each protein sample was separated using SDS-PAGE and transferred onto nitrocellulose membranes (Cat. 1620112, Bio-Rad, Hercules, CA). Next, the membranes were blocked with 5% BSA for 1 hour, and then incubated with primary antibodies against STING (Cat. 13647S, Cell Signaling), IRF3 (Cat. 4302S, Cell Signaling), p-IRF3 (Cat. 4947S, Cell Signaling), TBK1 (Cat. 3013S, Cell Signaling), p-TBK1 (Cat. 5483S, Cell Signaling), β-actin (Cat. 3700S, Cell Signaling), and Vinculin (Cat. 13901S, Cell Signaling) overnight at 4°C. The following day, the membranes were washed with PBST and incubated with anti-mouse (Cat. NXA931, GE Healthcare, Chicago, IL) or anti-rabbit (Cat. NA934V, GE Healthcare) secondary antibody for 2 hours before developing with ECL substrates (Cat. 170506, BioRad). The gel images were captured using Chem-DocXRS image acquisition machine (Bio-Rad).

The cells were scraped using ice-cold RIPA buffer in the presence of a protease and phosphatase inhibitor cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, MA, USA) and sonicated three times. Following incubation on ice for 30 min, the samples were centrifuged at 10,000× g for 20 min, and the supernatants were recovered and mixed with 2× Laemmli sample buffer and 2-mercaptoethanol (20%).
The Bradford colorimetric assay was used to determine the protein concentration and equal amounts of cell proteins (30 μg/lane) were separated by SDS-PAGE (10–12% polyacrylamide gel) and then electro-transferred onto nitrocellulose membranes by a Trans-Blot Turbo (Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Incubation was performed overnight at 4 °C with primary antibodies: rabbit polyclonal anti-occludin (Sigma Aldrich) and mouse monoclonal anti-actin (Santa-Cruz Biotechnologies, Dallas, TX, USA). HRP-conjugated secondary antibodies (1:3000; Bio-Rad, CA, USA) were incubated with the membranes for 1 h. The blots were then detected using an enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis was performed using Image Lab software 6.1.0. The results were expressed as arbitrary units (A.U.) and represented as the mean of three independent experiments.

Cell pellets were suspended in RIPA buffer (5 mM EDTA, 0.15 mol NaCl, 0.1 mol Tris pH 8.0, 1% Triton) with protease and phosphatase inhibitors cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and sonicated three times (for 10 s and 70% amplitude). The samples were incubated on ice for 30 min, and then centrifuged at 10,000× g for 20 min to remove pellet residues. Equal amounts of protein (30 μg/lane) estimated by Bradford protein assay were solubilized in a 2X Laemmli sample buffer containing 20% of 2-mercaptoethanol. Proteins were separated by SDS-PAGE on 10–12% polyacrylamide gel and then electro-transferred to nitrocellulose membranes (Trans-Blot Turbo Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Membranes were, then, incubated overnight at 4 °C with rabbit polyclonal anti-α-actin (Abcam, Cambridge, UK) and with mouse monoclonal anti-vinculin (Santa-Cruz Biotechnologies, Dallas, TX, USA). Thus, membranes were incubated for 1 h with HRP-conjugated secondary antibody (1:3000; Bio-Rad, CA, USA). The immune complexes were detected by enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis of the bands was performed using Image J software. The results were expressed as arbitrary unit (A.U.) and represented as mean of three independent experiments.

Whole cell lysates were prepared in RIPA buffer. Proteins were separated by SDS-PAGE under reducing conditions. BRCA1 protein was detected using mouse monoclonal anti-BRCA1 (SC-6954, 1:100, Santa Cruz Biotechnology). Protein bands were visualized using ECL substrates (BioRad, #170-5061) and imaged on Kodak X-OMAT 2000A.

Total proteins were purified in the absence of detergent [43 (link)]. Briefly, samples containing spores were pelleted by centrifugation and rinsed with 1× PBS. Total protein concentration was measured using the bicinchoninic acid assay (BCA) using protocols from the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoretically separated in 10% polyacrylamide-SDS gels using a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad^®, Hercules, CA, USA) [40 (link)]. The proteins were transferred to 0.45 μm nitrocellulose (Bio-Rad^®, Hercules, CA, USA) with a Trans-Blot Semi-Dry Transfer system (Bio-Rad^®, Hercules, CA, USA). Table 1 shows the tested antibodies. Following transfer, immunoblots were viewed with enhanced chemiluminescence (ECL) substrates (Bio-Rad^®, Hercules, CA, USA) using a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®, Marne-la-Vallee, France). Densitometry was conducted with FUSION FX software (Vilber^®, Marne-la-Vallee, France), using total protein to normalize measurements. Data represent three separate experiments from three different total protein isolations.

We followed the same procedures of Western blot analysis as previously described [9 (link), 38 (link)]. In brief, 72 h after drug treatment, we collected the cultured neurons from 6-well plates and extracted total protein with protein extraction buffer. Bradford assay was performed to measure protein concentration, and 30 μg of total protein was loaded for Bis-polyacrylamide gel electrophoresis (Bio-Rad). Electrophoresed proteins were transferred to nitrocellulose membrane (0.45 μm, Bio-Rad). The membranes were blocked with 5% non-fat milk in TBS-T at room temperature for 30 min followed by overnight 4 °C incubation with primary antibodies (rabbit anti-GFP, 1:1000, Novus Biologicals; rabbit anti-Ube3a, 1:1000, Bethyl Lab; mouse anti-actin; 1:5000, Sigma). The next day, the membranes were rinsed with TBS-T three times and incubated with HPR-conjugated secondary antibodies for 1 h at room temperature (goat anti-rabbit, 1:1000, Vector Lab or goat anti-mouse, 1:1000, Vector Lab). Following secondary antibody incubation, the membranes were rinsed with TBS-T at room temperature for 1 h (4–5 times) and ECL substrates (Bio-Rad) were used to visualize immunostaining using an Amersham Imager 600 (AI600, GE Life Sciences).

Mouse macrophages were isolated from BMMCs of 5TGM1/KaLwRij MM mouse models with CD11b MicroBeads (Miltenyi). Mouse macrophages were lysed in the mammalian cell extraction buffer (BioVision) with Protease and Phosphatase Inhibitors (ThermoFisher). Protein lysates were incubated on ice for 10 min and centrifuged at 14,000 × g for 10 min at 4 °C. Proteins were separated with 4–12% Bis-Tris Gel (Invitrogen) at 120 V for 1.5 h. And then transferred to a nitrocellulose (NC) membrane for 1.5 h at 200 mA. The membrane was blocked for 1.5 h with 5% milk at room temperature. Primary antibodies SHP-1 (Cell Signaling Technology, catalog # 3759, clone # C14H6, 1:1000), SHP−2 (Cell Signaling Technology, catalog # 3397, clone # D50F2, 1:1000), Phospho-SHP-1 (Tyr564) (Cell Signaling Technology, catalog # 8849, clone # D11G5, 1:1000), Phospho-SHP-2 (Tyr580) (Cell Signaling Technology, catalog # 5431, clone # D66F10, 1:1000), β-Actin (Cell Signaling Technology, catalog # 4967, 1:1000), were incubated overnight at 4 °C. Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, catalog # 7074, 1:1000) were incubated for 2 h. ECL Substrates (Bio-Rad, catalog # 1705060) were used for exposure. Western Blotting imaging was exposed with a Bio-Rad ChemiDoc XRS⁺. The images were analyzed by Image Lab Software (Bio-Rad).