β actin hrp

Western blot was performed as previously reported (27 (link)). The following antibodies were used for immunoblotting: OX40L (14991S), p105/p50 (13681S), p65 (6956S), RelB (10544S), c-Rel (67489S), HDAC1 (5356S), HSP90 (4877S), P-STAT3 (Tyr705, 9145S), STAT3 (12640S), P-STAT6 (Tyr641, 56554S), STAT6 (5397S), β-Actin-HRP (5125S), Goat anti-Rabbit IgG-HRP (7074S), Horse anti-Mouse IgG-HRP (7076S) from Cell Signaling Technology; p100/p52 (05–361) from Millipore; StarBright Blue 700 Goat anti-Mouse IgG (12004159), StarBright Blue 520 Goat anti-Rabbit IgG (12005870) from Bio-Rad; VeriBlot-HRP (ab131366) from Abcam. Images were acquired and analyzed using Bio-Rad ChemiDoc MP Imaging System.

Lysates were analyzed by SDS PAGE. Immunodetection was achieved using the following antibodies: LC3β (Cell Signaling, D11, 1:1000), RUBICON (Cell Signaling, D9F7, 1:1000), β-Actin HRP (Cell Signaling, 8H10D10, 1:10,000), and Anti-Rabbit IgG HRP (Cell Signaling, 1:10,000). Proteins were visualized by an ECL chemiluminescence reagent and imaged by a Protein Simple imager.

Neonatal microglia were isolated from P6-8 male and female mice using the same procedure as for adult microglia. Following Percoll gradient density centrifugation, cells from the same sex were pooled (up to 5 brains) and MACS-enriched using CD11b-biotinylated (clone M1/70, eBioscience) and MACS anti-biotin Microbeads (Miltenyi Biotec) following the manufactures recommended protocol. Enriched microglia (200,000 cells per condition) were stimulated with recombinant mouse IFNγ (rmIFNγ) as indicated in the figure legends. Cell lysates were prepared in 20 μL of RIPA buffer containing cOmplete Protease Inhibitor (Roche) and sodium orthovanadate on ice for 30 min. Lysates were run on a 10% SDS-PAGE gel and blotted for pSTAT1(Y701) (Cell Signaling Technology) and β-actin-HRP (Cell Signaling Technology). pSTAT1(Y701) bands were quantified using ImageJ and normalized to the respective β-actin band. Spleen homogenate from Cx3cr1^crex Usp18^fl/fl adult mice treated intraperitoneally with IFNβ for 24 h was used as a positive control for pSTAT1(Y701)^{72 (link)}.

Proteins were isolated from B cells using NP-40 lysis buffer and were resolved using NuPAGE 4–12% Bis-Tris gels (Thermo Fisher) and transferred to PVDF membranes using iBlot^™ 2 Gel Transfer system. Membranes were blocked with 5% non-fat dry milk in TBSTE buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween 20, 1 mM EDTA) and incubated with primary antibodies followed by appropriate secondary antibodies conjugated with horseradish peroxidase (HRP). Arid1a/BAF250A (1:1000, clone D2A8U) and β-actin HRP (1:5000, clone 13E5) were purchased from Cell Signaling.

Cells that were transfected with individual reporters were lysed with Lysis Buffer (50 mM Tris pH 7.8, 150 mM NaCl, 1% NP-40). Western blot analysis was performed, as described previously^{64 (link)}. The following primary antibodies were used in western analysis at the given dilution: GFP, 1:2000 (Clontech, 632381); β-actin-HRP, 1:2000, (Cell Signaling, 12262); Anti-mouse IgG HRP, 1:10,000 (Cell Signaling, 7076S).

Cell lysates were prepared in Pierce RIPA Buffer (Thermo Fisher Scientific, 89901) containing protease inhibitor and PhosStop cocktails (Roche, 5892970001 and 4906845001, respectively), separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane and probed. The following primary antibodies and dilutions were used: Strep Tag (Thermo Scientific, MA517282, 1:1000) and β-actin-HRP (Cell Signaling, 5125, 1:2000). Images were captured using the Azure c600 Western Blot Imaging System.