Ab183628

Cells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer for 30 min on ice. After centrifugation at 4°C for 10 min, the supernatant was removed, and the protein concentration was estimated calorimetrically using a Bio-Rad detergent-compatible assay. Protein samples (30 μg) were loaded onto SDS-polyacrylamide gels at a percentage appropriate for electrophoretic separation. Antibodies used for Western blotting were as follows: antibodies against EBNA3A (ab16126; Abcam), EBNA3B (clone 6C9; Allday lab), EBNA3C (clone A10; a gift from M. Rowe, University of Birmingham), EBNA1 (a gift from P. Farrell, Imperial College), EBNA2 (ab90543; Abcam), EBNA-LP (JF-186) (82 (link)), LMP1 (CS1-4; Dako), γ-tubulin (T6557; Sigma), RBPJ (J7A11-161; a gift from B. Kempkes, Helmholtz Zentrum München), KDM2B (09-864; Millipore), BMI1 (05-637; Millipore), SUZ12 (sc-46264; Santa Cruz), and mCherry (Ab183628; Abcam). In all blots, γ-tubulin was used as a loading control. The appropriate horseradish peroxidase (HRP)-conjugated antibodies were used as secondary antibodies (all from GE Healthcare). An ECL kit (Amersham Biosciences) was then used for visualization by autoradiography. In some cases, the membrane used for Western blotting was cut horizontally after protein transfer in order to facilitate multiple antibody probes and a single loading control for each blot used.

To verify expression of the MPODD::mCherry fusion protein, saponin pellets of schizont cultures from mpodd::mCh and mpodd(−) parasites were lysed in 50 µl RIPA buffer (50 mM Tris pH 8, 1% NP40, 0,5% sodium dexoycholate, 0,1% SDS, 150 mM NaCl, 2 mM EDTA). Probes were mixed and denaturated with Laemmli buffer and separated on 4–15% SDS–PAGE gels (Mini Protein TGX Gels, Bio-Rad). Gels were blotted on nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad) and incubated over night with antibodies directed against mCherry (rabbit polyclonal antibody, 1 : 5000 dilution, ab183628 from AbCam) or HSP70 (mouse monoclonal antibody, 1 : 5000 dilution, obtained through the MR4 as part of the BEI Resources Repository, NIAID, NIH: Mus musculus 65, MRA-662, MF Wiser). Secondary anti-rabbit (Immun-Star (GAR)-HRP, Bio-Rad) and anti-mouse antibodies (NXA931, GE Healthcare) conjugated to horseradish peroxidase were applied subsequently for 1 h (1 : 10 000 dilution). Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was conducted to identify Cse4 enriched regions in tagged species (K. bestiolae_mCherry-Cse4, K. europaea_mCherry-Cse4, and K. pini_mCherry-Cse4), using a polyclonal antibody against mCherry (ab183628, Abcam) as previously described [64 (link)]. Similarly, ChIP-seq for K. bestiolae CBS10118, K. europaea PYCC6329, K. pini CBS10737, K mangrovensis CBS8507, and K. dendrophila CBS6074 was performed to detect histone H3K9me2 enriched regions, employing a monoclonal antibody against histone H3K9me2 (ab1220, Abcam). Libraries were prepared and sequenced at the Duke University SGT, using either a NovaSeq 6000 or a HiSeq 4000 instrument to produce 50-bp paired-end reads. ChIP-seq sequencing reads were trimmed with Trim Galore v0.6.7, and subsequently aligned to each respective genome assembly with bowtie2. Read duplicates were removed with Picard and SAMtools, and bamCompare v3.5.4 was used to normalize the ChIP-seq data against the input control. The resulting files were converted to bedGraph format for visualization in IGV or for plotting with pyGenomeTracks.

GBP1^KO cells stably transduced with pInducer-GBP1 constructs were incubated overnight with cell culture medium containing the concentrations of aTc indicated in Fig. 6 and 7. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1× protease inhibitor cocktail (Sigma) for 5 min on ice. Lysates were spun for 10 min at maximum speed in a microcentrifuge at 4°C and then combined with an equivalent volume of 2× Laemmli sample buffer containing β-mercaptoethanol and incubated at 95°C for 10 min. Samples were run on 4 to 15% Mini-Protean TGX gels (Bio-Rad) and transferred to nitrocellulose. Immunoblots were probed with 1:1,000 anti-GBP1 (Abcam, Inc.; ab131255), 1:1,000 anti-mCherry rabbit polyclonal (Abcam, Inc.; ab183628), or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit polyclonal (Abcam, Inc.; ab9485) antibody, followed by horseradish peroxidase-conjugated anti-rabbit IgG (Invitrogen).

Immunofluorescent staining was performed as previously reported.¹⁶ Mice were anesthetized with 2% isoflurane and perfused with 0.9% saline, followed by 4% paraformaldehyde. The whole brains were rapidly isolated and fixed in 4% paraformaldehyde overnight and sliced into 10 μm‐thick frozen sections at the vHPC and mPFC levels. The frozen sections were washed 3 times for 10 min in phosphate‐buffered saline (PBS) and then incubated with blocking buffer (3% BSA in PBS, 0.1% Triton ×100) for 50 min. The sections were subsequently incubated at 4°C overnight with the following primary antibodies: anti‐mCherry (1:500, ab183628, Abcam) and anti‐c‐Fos (1:500, ab208942, Abcam). On the following day, the sections were washed 3 times with PBS and incubated with the corresponding secondary antibodies diluted in PBS (1:500) for 60 min. The secondary antibodies included Alexa Fluor 488 (ab150113, Abcam) and Alexa Fluor 594 (ab150080, Abcam). After washing 3 times in PBS, the sections were re‐stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI) and analyzed under a fluorescence microscope. Image J software was used to count the number of c‐Fos‐positive cells. Fluoro‐Gold was directly detected and photographed with ultraviolet light (380 nm excitation wavelength, 420 nm absorption wavelength) under a fluorescence microscope.