Rabbit anti tubulin

For immunoblots, 20 μg of TBS-soluble or TBSX-soluble brain samples and Laemmli buffer with 10% β-mercaptoethanol were heated to 95°C for 5 min. Samples were subjected to gel electrophoresis and separated by size (Western blot) or isoelectric point and size (Isoelectric Focusing). Separated proteins were transferred to nitrocellulose (Western blot) or PVDF (Isoelectric Focusing) membranes and incubated in blocking buffer (5% non-fat dry milk in TBS with 0.05% Tween 20 (TBS-T)) for 1 hr. The following primary antibodies were diluted 1:1000 in blocking buffer and incubated overnight at 4°C: mouse anti-APOE (D6E10, ab1906, Abcam, Cambridge, MA, USA), mouse anti-IκB-α (L35A5, 4814, Cell Signaling, Danvers, MA, USA), rabbit anti-COX2 (ab15191, Abcam, Cambridge, MA, USA), rabbit anti-tubulin (Sigma, St. Louis, MO, USA), mouse anti-β-actin (Sigma, St. Louis, MO, USA). Membranes were washed 3× in TBS-T. The appropriate horseradish peroxidase-conjugated secondary antibody was diluted 1:5000 in blocking buffer and incubated at room temperature for 1 hr. Membranes were then washed 3× in TBS-T. SuperSignal West DURA luminol/enhancer solution (Pierce, Rockford, IL, USA) was added to membranes for 5 min. The density of bands was quantified using ImageJ software (NIH, Bethesda MD, USA).

Protein extracts and immunoprecipitates were run on 10% SDS-polyacrylamide gels and transferred to PVDF Immobulon membranes (Thermo). The following antibodies were used: mouse anti-HA (Roche), mouse monoclonal anti-DDX41 and goat anti-LaminB (SantaCruz Biotechnology), rabbit anti-STING and anti-FLAG and HRP-conjugated anti-rabbit (Cell Signaling), mouse anti-V5 (Invitrogen), rabbit anti-tubulin (Sigma), HRP-conjugated anti-goat and -mouse antibody (Sigma). ECL kits (GE Healthcare Life Sciences) or Supersignal West Femto Chemiluminescent substrate (Thermo Scientific) were used for detection.