Infrared scanner

Mouse cortical proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Solarbio, China). The protein concentration was quantified by a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, USA). Then, equivalent amounts of protein (50 μg) with buffer were loaded in gels and separated by SDS-PAGE. Next, proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Roche, USA) and blocked with 5% nonfat milk for 1 h at room temperature, followed by incubation with primary antibodies specific for occludin (Invitrogen, US), claudin5 (Bioworld Technology, USA), TNFα (Santa Cruz, USA), Histone H3 (Abcam, UK), citH3 (citrulline R2+R8+R17) (Abcam, UK), and β-actin (Cell Signaling Technology, USA) overnight at 4°C. After being washed three times with TBST, the membrane was incubated with anti-rabbit IgG secondary antibody (Rockland, USA) or anti-mouse IgG secondary antibody (Rockland, USA) for 1 h at room temperature and then was washed with TBST for another three times. Finally, the membrane was scanned by an infrared scanner (LICOR Bioscience, USA). The intensity of the bands was calculated by the ImageJ software.

The harvested cells were washed twice with cold PBS, then lysed in 500 μl RIPA buffer [150 mM NaCl, 50 mM Tris (pH 7.6), 1% Nonidet P-40, 2 mM EDTA, proteinase inhibitor mixture (1 mM phenmethyl sulfonyl fluorine, 1 g/ml aprotinin, 1 g/ml leupeptin, and 1 g/ml pepstatin)] for 30 min with constant oscillation at 4 °C. After centrifugation at 14,500 rpm, 4 °C for 5 min, cell debris was removed. The supernatant was then transferred to a new eppendorf tube, after which the protein samples were boiled in 6x DTT loading buffer according to the concentration of protein that was determined by the Bradford method. Next, the samples were separated by SDS-PAGE and then transferred to a 0.45-mm nitrocellulose filter. After blocking, the protein in the membrane was probed with primary antibodies at 4 °C overnight, and then incubated with appropriate secondary antibodies for another 1 h at room temperature. Finally, the membrane was visualized using an Odyssey Infrared scanner (Li-Cor Biosciences, US) for optical density analysis.

Peptide libraries were produced by automatic SPOT synthesis (51 (link)). They were synthesized on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc (9-fluorenylmethyloxycarbonyl) chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments, Berlin) (52 (link)). The SNAP25 and CSPα arrays consisted of 15-mer peptides (200 peptides each), with all possible amino acid substitutions within a 10-amino acid stretch (sequences and serial substitution shown in Fig. 1). The dried membranes were submerged in 100% ethanol for 2 min, washed briefly with distilled water, and then incubated with blocking buffer: 5% (w/v) milk in PBS-T (PBS containing 0.02% Tween) for 2 h at room temperature. Then, after a brief washing with PBS-T, overnight incubation with the ARzD17-His protein (500 nm in PBS-T) took place at 4 °C. Membranes were then washed extensively with PBS-T, incubated with mouse His₆ tag antibody (1/2,000 dilution in PBS-T) for 1 h at room temperature, washed again with PBS-T, incubated with mouse secondary antibody (1/10,000 dilution in PBS-T), and after extensive washes with PBS-T containing 0.2% Tween, ARzD17-His-bound peptides were visualized using a LI-COR infrared scanner. Spots were quantified using Image Studio software (version 2.0), with an area of the blot containing no peptide assigned as background.

For co-immunoprecipitation, eluted products and 10% of the input were separated by SDS-PAGE and transferred to a PVDF membrane for blotting at 4C with Gli3 (polyclonal goat IgG 1:1000, R and D Systems) and Hand2 (polyclonal goat IgG or mouse monoclonal IgG₁ 1:1000) primary antibodies. Detection of primary antibodies was performed using infrared-conjugated secondary antibodies (donkey anti-goat or goat anti-mouse IRDye 800CW, LICOR) and acquired using a LICOR infrared scanner. For plasmid verification, F primary antibody (monoclonal M2 mouse IgG₁) and enhanced chemiluminescence assay (Amersham ECL Primer, GE Healthcare Life Science) were used for detection.

For cells sorted from co-culture experiments, total RNA was extracted using the Direct-Zol^RM RNA MiniPrep Plus (Zymo Research) according to the manufacturer’s protocol. For isolated cells from experimental mice, RNA was extracted using the Quick-RNA microprep kit (Zymo Research) according to the manufacturer’s protocol. Reverse transcription was carried out using the RevertAid RT kit (Thermo Fisher) following manufacturer’s protocol. Primer sequences used are listed in Supplementary Tables 1 and 2. Reactions were performed using Powerup SYBR Green Master Mix (Applied Biosystems) using QuantStudio 6 Flex. Relative gene expression was calculated by ddCT method normalized to β-actin on the QuantStudio Real-Time PCR Software v.1.2 (Applied Biosystems).
For western blot, total protein lysate was extracted from astrocytes using RIPA lysis buffer containing protease (Roche) and phosphatase inhibitors (Thermo Fisher). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were washed and blocked with Odyssey blocking buffer (Li-Cor) and stained for primary antibodies overnight at 4 °C. The membranes were washed and probed with secondary antibodies for 2 h in the dark at room temperature. Blots were scanned using an infrared scanner (Li-Cor).