Rat monoclonal anti ha 3f10

The bloodstream forms of T. brucei were washed with PBS and resuspended and fixed in PBS containing 4% paraformaldehyde for 5 min at room temperature. The fixed parasites were washed with PBS and allowed to attach to coverslips for 5 min. The coverslips were submerged in PBS containing 0.1% Nonidet P40 for 5 min to permeabilize the cells. Subsequently, PBS containing 3% bovine serum albumin was added for a 1-h blocking period. After blocking, the cover slips were incubated for 1 h with a 1:1000 dilution of rabbit polyclonal anti-TbGRASP and 1 μg/mL of rat monoclonal anti-HA 3 F10 (Roche Diagnostics) in CanGetSignal Immunostain A (TOYOBO), followed by three 10-min washes in PBS containing 0.5% BSA. Following this, the cover slips were washed thrice in PBS containing 0.5% BSA and incubated for 1 h with a 1:1000 dilution of AlexaFluor 594-conjugated anti-rabbit IgG (Life Technologies) and 2 μg/mL of AlexaFluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Danvers, MA) in CanGetSignal Immunostain B. The coverslips were subsequently washed thrice and mounted in antifade mounting solution containing DAPI (Vector Laboratories). Images were obtained with a confocal laser scanning microscope 510 (Zeiss). All images were obtained and processed under the similar settings.

Ovaries were dissected in PBS, followed by fixation in 4% paraformaldehyde in PBS for 20 min at room temperature. After washing with PBST (PBS + 0.2% Triton X-100) twice, the ovaries were permeabilized in PBST-5 (PBS + 0.5% Triton X-100 with 5% goat serum). After washing once with PBST-B (PBS + 0.2% Triton X-100 + 1% BSA), the ovaries were incubated with primary antibody (1:150, rat monoclonal anti-HA: 3F10, #11867423001, Roche, Indianapolis, IN, USA and rabbit anti-Ago3 (1:1000) (Brennecke et al., 2007 (link))) in PBST-B at 4 °C overnight. Washing was performed in PBST 3 times, 2 min each at room temperature, followed by 20 min wash in PBST 3 times and 2 min wash in PBST-B once at room temperature. The ovaries were incubated with secondary antibody Alexa Fluor 488 goat anti-rat IgG (H+L) (1:100, #A-11006, Life Technologies) in PBST for 2 h at 4 °C. After secondary antibody incubation, washing was performed in PBST three times, 2 min each at room temperature, followed by 20 min wash in PBST three times and 2 min wash in PBST-B once at room temperature. After washing, the ovaries were carefully separated on slides by fine forceps and mounted with Vectashield mounting medium with DAPI (#H-1200; Vector laboratories, Burlingame, CA, USA).

Mission shRNA against ERβ was obtained from Sigma–Aldrich. Expression plasmid for HA-tagged mERβ (pSSH25-mERβ) was constructed by inserting PCR-amplified mERβ cDNA into pSHH25 [56 (link)] using XhoI and BlgII restriction sites. pSSH25-TDG for mammalian expression of HA-fused TDG [56 (link)], pPRS220 for yeast expression of Gal4-AD-fused TDG [46 (link)], 3× ERE-luc [47 (link)], and pSG5hERβ [57 (link)] used in luciferase assays have been published. pRL-TK for normalisation of luciferase activity was purchased from Promega. GST-ERβ was constructed by cloning cDNA encoding for human ERβ into pGEX-6P-3 (GE Healthcare) using BamHI and XhoI restriction sites. pACT2-ERβ was obtained by cloning cDNA encoding for human ERβ into pACT2 (Clontech) using SmaI and XhoI restriction sites. Antibodies used were as follows: rat monoclonal anti-HA (3F10) from Roche Applied Science, rabbit monoclonal anti-ERβ (05-824), rabbit polyclonal anti-H3K4m2 (07-030), and rabbit polyclonal anti-H3K27m3 (07-449) from Millipore, rabbit polyclonal anti-H3K9m3 (060-050) from Diagenode, and mouse monoclonal anti-Hsp90 (F-8) from Santa Cruz Biotechnology, Inc.

Rat monoclonal anti-HA (3F10, 1:1,000) and mouse monoclonal anti-GFP (B-2, 1:1,000) antibodies were purchased from Roche and Santa Cruz Biotechnology, respectively. Mouse monoclonal anti-H3 (1B1-B2, 1:5,000) was obtained from Active Motif. Rabbit polyclonal anti-Myc (ab9106, 1:2,000, Abcam) or mouse monoclonal anti-Pol II-S5P (3E8, generated by the lab of D. Eick) antibodies were kindly provided by M. Spletter and by A. Ladurner, respectively. Mouse monoclonal H3K9me2 ChIP grade (ab1220) was purchased from Abcam. Secondary antibodies fused to HRP were used for detection (goat anti-mouse HRP 1:3,000, BioRad; goat anti-rat HRP 1:3,000, Merck Millipore; goat anti-rabbit HRP 1:3,000, BioRad)

The bloodstream forms of T. brucei were washed with PBS and resuspended and fixed in PBS containing 4% paraformaldehyde for 5 min at room temperature. The fixed parasites were washed with PBS and allowed to attach to coverslips for 5 min. The coverslips were submerged in PBS containing 0.1% Nonidet P40 for 5 min to permeabilize the cells. Subsequently, PBS containing 3% bovine serum albumin was added for a 1-h blocking period. After blocking, the cover slips were incubated for 1 h with a 1:1000 dilution of rabbit polyclonal anti-TbGRASP and 1 μg/mL of rat monoclonal anti-HA 3 F10 (Roche Diagnostics) in CanGetSignal Immunostain A (TOYOBO), followed by three 10-min washes in PBS containing 0.5% BSA. Following this, the cover slips were washed thrice in PBS containing 0.5% BSA and incubated for 1 h with a 1:1000 dilution of AlexaFluor 594-conjugated anti-rabbit IgG (Life Technologies) and 2 μg/mL of AlexaFluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Danvers, MA) in CanGetSignal Immunostain B. The coverslips were subsequently washed thrice and mounted in antifade mounting solution containing DAPI (Vector Laboratories). Images were obtained with a confocal laser scanning microscope 510 (Zeiss). All images were obtained and processed under the similar settings.

CpG oligodeoxynucleotide-1826 (CpG DNA) and biotin-CpG DNA were purchased from TIB Molbiol (Germany). R848 and LPS (Escherichia coli O111:B4) were from Enzo Life Sciences (USA) and Sigma-Aldrich (USA), respectively. Rat monoclonal anti-HA (3F10) was purchased from Roche (USA). Mouse monoclonal anti-GFP (B-2) was purchased from Santa Cruz (USA). Mouse monoclonal anti-myc (9B11), rabbit monoclonal anti-MyD88 (D80F5), mouse monoclonal anti-phospho-Iκβ (5A5), and anti-Iκβ were obtained from Cell Signaling Technology (USA). Rat anti-LAMP1 (1D4B) was obtained from BD Biosciences (USA). Rabbit anti-actin was obtained from Bethyl Laboratories (USA). Rabbit anti-GFP and rabbit anti-myc antibodies were made in our laboratory by immunizing rabbits with purified GFP protein and Myc epitope tag peptide (EQKLISEEDL), respectively.