Phospho tbk1 ser172

Human monocytes were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). Lysates were separated by NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 50% Odyssey Blocking buffer in PBS-T (0.1% Tween 20 in PBS) and incubated with appropriate antibodies overnight at 4°C. The following antibodies were used: STAT1, phospho-STAT1(Y701), STAT2, phospho-STAT2 (Y690), TBK1, phospho-TBK1 (Ser172), LAMP1 (D2D11) XP, SQSTM1/p62, phospho-S6 (Ser235/236), GAPDH, and β-actin (Cell Signaling Technology); IRF3 and phospho-IRF3 (S386) (Abcam); and LC3 and vinculin (Sigma-Aldrich). Antibody suppliers and catalog numbers are shown in Supplemental Table 10. Primary antibody incubations were followed by incubation with IRDye secondary antibodies for 1 hour at room temperature. Immunoblots were imaged using Odyssey CLx Imaging System (LI-COR Biosciences). Protein band intensity was quantified using ImageJ (NIH) and normalized to β-actin or vinculin. Detailed information about antibodies used in this study is summarized in Supplemental Table 10.

Frozen cell pellets where lysed and supernatants prepared and immunoblots prepared as described previously [11 (link)]. Membranes were probed with antibodies to detect IRF-3(D83B9;Cat# 4302), phospho-IRF-3 (Ser396; Cat#29047), STING (D2P2F; Cat#13647), phospho-STING (Ser366; Cat#19781), TBK1 (D1B4; Cat#3504), phospho-TBK1 (Ser 172; Cat# 5483), NF-kappa B p65(D14E12; Cat# 8242), phospho-NF-kappa B p65 (Ser536; Cat# 3033), cleaved PARP1(Asp214; Cat#9541), phospho-Histone H2A.X (Ser139; Cat#2577) from Cell Signalling (Arundel, QLD, Australia) and PD-L1 (Roche, Sydney, NSW Australia).

The following antibodies were used: mouse monoclonal antibodies to α-tubulin (Santa Cruz, sc23948), GAPDH (Santa Cruz, sc32233), phospho-H2A.X (Ser139) (EMD Millipore, 05-636), anti-γ-tubulin (Sigma-Aldrich, T5326), and IRF3 (Abcam, ad50772), which were used at a 1:1000 dilution in phosphate-buffered saline (PBS) with 3% bovine serum albumin (BSA); rabbit monoclonal antibodies to cGAS (Cell Signaling, #15102S), STING (Cell Signaling, 13647), phospho-IRF3 (Ser386) (Abcam, ab76433), phospho-IRF3 (Ser396) (Cell Signaling, #29047S), phospho-TBK1 (Ser172) (Cell Signaling, 5483S), and TBK1 (Cell Signaling, 3504S), which were used at a 1:1000 dilution in PBS with 3% BSA. Horseradish-peroxidase-conjugated anti-mouse (G21040) and anti-rabbit (G21234) antibodies were obtained from Invitrogen (at a 1:3000 dilution in TBST). The following fluorochrome-conjugated secondary antibodies were used at a 1:500 dilution in PBS with 3% BSA: anti-mouse Alexa-488 (Invitrogen, A11059), anti-rabbit Alexa-488 (Invitrogen, A11034), and anti-mouse Cy3 (Jackson ImmunoResearch, 715-165-151).

The following antibodies were used: rabbit antibodies specific for IRF3, phospho‐IRF3 (Ser396), TBK1 and phospho‐TBK1 (Ser172) were purchased from Cell Signaling Technology Inc. Anti‐α‐Tubulin antibody was obtained from Santa Cruz Biotechnology and anti‐fibrillarin antibody was from Abcam. The following secondary antibodies, anti‐rabbit‐horseradish peroxidase (HRP) and anti‐mouse HRP were from Jackson Immuno Research Laboratories. Goat anti‐Rabbit IgG (H+L) Highly Cross‐Adsorbed Secondary Antibody‐Alexa Fluor 488, Alexa Fluor 647 and Goat anti‐Mouse IgG (H+L) Highly Cross‐Adsorbed Secondary Antibody‐Alexa Fluor 488, Alexa Fluor 568 were purchased from Thermo Fisher Scientific.
PI3K‐specific inhibitors, Pictilisib (GDC‐0941) and ZSTK474, were obtained from Selleck Chemicals. AKT inhibitor, Akti1/2, was got from Sigma.

Cells were lysed with RIPA (Millipore Sigma, Cat# 20-188) containing protease inhibitor (Merck, Cat#11836170001) and phosphatase inhibitor (Sigma Aldrich, Cat# 04906845001). Protein gels were transferred to polyvinylidene difluoride membrane using iBlot two instrument and consumables (Thermo Fisher Scientific, Cat# IBI21001, IB24001). Membranes were blocked with 5% BSA for 1 h, primary antibodies were incubated overnight at 4 °C, and appropriate secondary antibodies (LI-COR Biosciences, Cat# 926–32211, 926–68070) were stained for 1 h at room temperature. Blots were imaged using LI-COR Odyssey CLx scanner and processed using Image Studio Lite. Signal for OAS2 knock-in MCF10A AKT cells was too low to quantify by fluorescence and was instead imaged using HRP chemiluminescence (Cell Signaling Technology, Cat# 7076S and 7074S).
The following antibodies were used at recommended manufacturer dilutions: phospho(Ser172)-TBK1 (Cell Signaling Technology, Cat# 5483T), TBK1(Cell Signaling Technology, Cat# 3504T), ACE2 (Cell Signaling Technology, Cat# 4355T), TMPRSS2 (Invitrogen, Cat# MA5-35756), OAS2 (Thermo Fisher Scientific, Cat# TA802770), MYC (Cell Signaling Technology, Cat# 9402S), and ACTINβ (Cell Signaling Technology, Cat# 3700S, 4970S).