Protein a beads

After treatment protocol, hCMEC/D3 were washed with PBS and lysed with RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40, Boston Bioproducts, BP-115) together with phosphatase /protease inhibitor cocktail (Cell Signaling, 5872S) for 20 min. After mixing, 10 μL Protein A beads (Santa Cruz, CA) with 5 μg P-Thr (H-2) (Santa Cruz, sc-5267) or P-Tyr (PY99) antibody (Santa Cruz, sc-7020) for 2 h, 200 μL lysates were diluted with 1xPBS buffer to 1 mL, and followed by addition of the pre-mixed antibody-protein A mixture. After overnight incubation at 4°C, beads were washed with 1x PBS for 3 times and centrifuged at 8000 rpm, 2 min at 4°C. Finally, beads were suspended in 60 μL 1x PBS, and SDS-loading buffer was added to denature the protein sample. The precipitated proteins were resolved by Western blot.

Media containing GP1-Fc were incubated with Protein A beads (Santa Cruz) for 1 hour at 4°C and washed 3 times with NETI buffer (50 mM Tris-HCl, 150 mM sodium chloride, 1 mM EDTA, 0.5% (vol/vol) IGEPAL). HEK293T cell extracts were prepared in NETI, adjusted to pH 5.0, and incubated for 1 hour at 4°C with the GP1-Fc beads. Proteins were eluted using TBS buffer and precipitated using acetone at -20°C. Pellets were recovered in sample buffer for SDS-PAGE and immunoblot analysis. Anti-LAMP1 antibody (CD107a) was obtained from Millipore and anti-human IgG from Abcam.

HeLa or H1299 cells were lysed in immunoprecipitation (IP) buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing protease inhibitor cocktails. The cell lysates were incubated with mouse anti-Myc (CST, 2276) or rabbit anti-poly/mono-ADP Ribose (E6F6A) monoclonal antibody (CST, 83732) on a rotary shaker at 4 °C overnight. Mouse or rabbit IgG (Santa Cruz, sc-2025 or sc2027) was used as a negative control for detecting the pull-down specificity. Protein G beads (Santa Cruz, sc-2001) or protein A beads (Santa Cruz, sc-2002) were added and incubated at room temperature for 2 h. The agarose beads were collected by centrifugation, washed five times with IP buffer according to the manufacturer’s instructions, and eluted in SDS sample buffer for the subsequent Western blotting assay. Olaparib (Selleck, S1060) was purchased from Selleck Chemicals. The instrument used for X-ray irradiation was a RAD SOURCE RS 2000pro-225 X-RAY IRRADIATOR.

After fixation in 1 % formaldehyde, T cells (1 × 10⁷) were lysed for 5 min in 50 mM Tris buffer (pH 8) containing 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 % Nonidet P (NP)-40 and 10 % glycerol supplemented with anti-protease cocktail (Roche Life Science, Indianapolis, IN, USA). Nuclei were resuspended in 50 mM Tris buffer with 1 % SDS and 5 mM EDTA. Chromatin was sheared by sonication. After preclearing with protein A beads (Santa Cruz Biotechnology), lysates were incubated overnight at 4 °C with 1 μg/ml anti-GATA-3 (sc-269) or mouse immunoglobulin G (IgG) antibodies (Santa Cruz Biotechnology). Immune complexes were collected with protein A, washed three times with high-salt buffer (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % sodium deoxycholate), twice with low-salt buffer (10 mM Tris–HCl, 250 mM LiCl, 1 mM EDTA, 0.5 % NP-40, 0.1 % sodium deoxycholate) and then twice with Tris-EDTA (TE). Immune complexes were extracted in 1× TE buffer, and protein crosslinking was reverted by heating at 65 °C for 5 h. DNA was then extracted by phenol-chloroform and precipitated by ethanol, and a 1/20 fraction of the immunoprecipitated DNA was used for qRT-PCR.