Anti gfp sc 8334

The protein concentrations of cell lysates were estimated by the bicinchoninic acid (BCA) protein assay reagent (Pierce) using BSA as a standard. Cell extracts from HEK293T cells (60 μg) or dechorionated and deyolked larvae at 3 dpf obtained using 200 μl lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40 and a 1:20 dilution of the protease inhibitor cocktail P8340 from Sigma-Aldrich) were resolved on 10% SDS-PAGE, transferred for 50 min at 200 mA to nitrocellulose membranes (BioRad), probed with a 1/200 dilution of four different mAbs to zebrafish Gbp4 generated using the SEAL technology (Abmart; Supplementary Fig. 1), with a 1/5,000 dilution of the mouse monoclonal Ab against c-myc (46-0603; Invitrogen) or 1/1,000 dilution of the rabbit polyclonal anti-GFP (sc-8334; Santa Cruz), and developed with adequate HRP labelled secondary antibodies or with enhanced chemiluminescence (ECL) reagents (GE Healthcare) according to the manufacturer's protocol. In some experiments, membranes were then reprobed with a 1/5,000 dilution of a commercial rabbit antibody to histone 3 (#ab1791, Abcam), as an appropriate loading control. Images have been cropped for presentation in Figs 1 and 6. Full size images are presented in Supplementary Fig. 14.

Embryos were dechorionated and fixed for 20 minutes in 10% formaldehyde overlayed with n-heptane at room temperature. Devitellinisation was achieved by adding chilled methanol and shaking vigorously. Embryos were washed twice with methanol before rehydrating in a step-wise manner with PBS and blocked with 4% normal goat serum. Imaginal discs from third instar larvae were fixed in 4% formaldehyde for 20 minutes before blocking.
Immunostaining of embryos was performed over night with anti-H B (directed against C-terminal fragment of H) and of wing discs with anti-H A (directed against central H fragment)22 (link), with anti-Phospho-Histone H3 (Ser28) (#9713, Cell Signaling Technology, Massachusetts, USA), and anti-GFP (sc-8334, Santa Cruz Biotechnology, Inc., Texas, USA). Secondary antibodies fused to FITC, Cy3 and Cy5 respectively were used (Jackson ImmunoResearch, Pennsylvania, USA). Embryos and imaginal tissue were mounted in VectaShield (Vector Laboratories, California, USA) before being imaged on a Bio-Rad MRC1024 system running LaserSharp 2000 imaging software (Bio-Rad Laboratories, California, USA) coupled to a Zeiss Axioskop (Carl Zeiss AG, Oberkochen, Germany). Figures were assembled using Corel Draw and Photo Paint software.

Human 293T, THP-1, and mouse macrophage RAW 264.7 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic. Mouse BMDMs were generated by flushing bone marrow cells from the femurs and tibias of mice and were maintained in DMEM containing 10% FBS and 10% conditioned media from L929 cells overexpressing macrophage colony-stimulating factor (M-CSF). Poly(I:C), poly(dAdT), and LPS were from InvivoGen (San Diego, CA). Anti-MAVS (3393), anti-TRIF (4596S), anti-FOSL1 (5281), anti-TBK1 (3013), and anti-p-IRF3 (4947) were from Cell Signaling Technology, Inc. (Danvers, MA); Anti-K63 (05-1308) and anti-K48 (05-1307) were from EMD Millipore; anti-IRF3 (sc-9082), anti-TRAF3 (sc-6933), anti-FOSL1 (SC-605), and anti-GFP (sc-8334) were from Santa Cruz Biotechnology (Dallas, TX); anti-MYC-horseradish peroxidase (anti-MYC-HRP) (11814150001) was from Roche Applied Science (Indianapolis, IN); and anti-FLAG-HRP (M2) and anti-β-actin (A1978) were from Sigma (St. Louis, MO). Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate antibody (A-11034) was from Thermo Fisher Scientific. Protein G agarose used for immunoprecipitation was from Santa Cruz Biotechnology.

Cells were lysed in immunoprecipitation lysis buffer (Thermo Fisher Scientific) supplemented with a complete protease inhibitor cocktail (Roche). Co-IP was performed using the SureBeads Protein A magnetic beads (Bio-Rad Laboratories) according to the manufacturer’s instructions. SureBeads were conjugated with 1 μg antibodies for 15 min. The following antibodies were used; anti-GFP (SC-8334) from Santa Cruz Biotechnology, anti-HA (51064-2-AP) and anti-MYC (16286-1-AP) from Proteintech, anti-STING (CST#13647) from Cell Signaling Technology, anti-MAVS (A300-782A) was purchased from Fortis Life Science and anti-LC3 (NB100-2220) from Novus. After antibody conjugation, 500 μg cell lysate was added, and incubated overnight at 4 °C. Immunoprecipitates were washed with 0.1% PBS/Tw three times, and proteins were eluted in SDS sample buffer and analyzed by immunoblot anaylsis to detect endogenous interactions between proteins. To examine ubiquitinated Parkin, lysed cells were incubated with magnetic beads which were conjugated with Parkin antibody (CST#4211) for 24 h and then immunoprecipitates were analyzed by immunoblot assay using phospho-ubiquitin (CST#62802).