Normal mouse igg

Magna Bind goat anti-mouse IgG magnetic bead slurry, 100 μL, (Thermo Scientific, Waltham, USA) was incubated with 10 μg of mouse monoclonal anti-Ago2 (Abcam, Cambridge, UK) or mouse normal IgG (Santa Cruz Biotechnology, Dallas, US) antibodies for 2 h at 4 °C. The antibody-coated beads were then added to plasma and incubated overnight at 4 °C with rotation. Beads were washed and each sample then eluted in RNAse free water before QIAzol was added for RNA isolation. Ago2 isolation was determined by western blot analysis.

Immunohistochemical staining was performed as described previously [16 (link)]. Briefly, specimens were fixed with 3.7% formalin and embedded in paraffin, and then sliced sequentially at a thickness of 3 μm. Samples were deparaffinized with xylene and rehydrated with graded alcohol. After rinsing with tris-buffered saline, samples were processed for antigen retrieval with EDTA buffer (pH 9.0) at 95 °C for 20 min. Samples were incubated with antibodies against Arf6 (1:100) or AMAP1 (1:200), or normal mouse IgG (1:100) at room temperature for 60 min, and the Histofine Simple Stain MAX PO system (Nichirei) was used for visualization. The coloring reaction was performed with 3,3′-diaminobenzidine (Dojin) for 5 min. Hematoxylin was used as a counterstain. A rabbit polyclonal antibody against Arf6 and mouse normal IgG was purchased from commercial sources (Arf6, Aviva; mouse normal IgG, Santa Cruz Biotechnology, Inc.).

RIP assay was performed using cell extracts from mock- or IAVΔNS1- infected HeLa cells with anti-human DHX36 monoclonal antibody by RiboCluster Profiler RIP-Assay Kit (MBL, Japan, RN1001) according to the manufacturer's recommendations. Briefly, RNA-protein complex was pulled-down with 5 µg of mouse normal IgG (Santa Cruz, sc-2025) or anti-DHX36 monoclonal antibody. Then, bound RNAs were recovered from the RNA-protein complex and used for cDNA synthesis. Real-time qPCR was further performed to evaluate the RNA level bound to DHX36 with the specific primer sets targeting IAV gene. As an internal control, human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted. Sequence information of primers is as follows: IAV segment 5, 5′-GATGGAGACTGATGGAGAACGCCAG-3′ (Sense), 5′-AGCTGTTTTGGATCAACCGTCCCTC-3′ (antisense); IAV segment 6, 5′-GGACAGAGACTGATAGTAAG-3′ (sense), 5′-GTTAGCTCAGGATGTTGAAC-3′ (antisense); IAV segment 8, 5′-GATAACACAGTTCGAGTCTC-3′ (sense), 5′- TTTCTCGTTTCTGTTTTGGA-3′ (antisense); human GAPDH, 5′-ACTGCCAACGTGTCAGTGGT-3′ (sense), 5′-TTACTCCTTGGAGGCCATGT-3′ (antisense).

Immunoprecipitation was performed using total protein extracts from respective cells in various conditions (~300 mg), together with 1 mg of respective antibodies (as indicated in the figures). After overnight incubation at 4 °C, immune complexes were precipitated together with protein A-Sepharose beads (Amersham Biosciences) and analyzed by immunoblotting as described above. RIP assay was performed using total protein extracts from mock- or viral-infected cells with indicated antibody by RiboCluster Profiler RIP-Assay Kit (MBL, Japan, RN1001) according to the manufacturer’s recommendations. In brief, RNA–protein complex was pulled down either with 5 mg of mouse normal IgG (#sc-2025, Santa Cruz Biotechnology) or antibodies of interest. The bound RNAs were then recovered from the RNA–protein complex and used as a template for cDNA synthesis. Reverse-transcription and quantitative-PCR (RT-qPCR) was further performed to evaluate the RNA level bound to respective pulled-down proteins with the specific primer sets targeting the respective viral-associated gene. As an internal control, the glyceraldehyde 3-phosphatedehydrogenase (GAPDH) gene was used. Northern blotting was performed as previously described [13 (link)]. RNA probes to detect NDV F-mRNA was prepared from NDV cDNA using primer sets: Forward 5′-GCACAGATAACAGCAGCCTC-3′ and reverse 5′-CATCTTCCCAACTGCCACTG-3′.

A549 cells were cross-linked with 1% formaldehyde at RT for 10 min. The cross-linked chromatin was sonicated to generate DNA fragments averaging 100–200 bp in length by Bioruptor plus. Chromatin fragments were immunoprecipitated with antibodies against mouse normal IgG (4 μg, Santa Cruz), BARX1 (4 μg, Santa Cruz) and protein A-Sepharose beads. After washing and reversing the cross-links, the enriched DNA was purified and then examined by qRT-PCR. The primer sequences are listed in Supplementary Table 2.

Transfected 293T cells or testes dissected from 1-year-old adult X.
laevis were homogenized in RIPA buffer, followed by sonication. The
cell extracts from a 35 mm dish with 1 μg of each anti-DMRT1 monoclonal
antibody, or the testicular extracts (10 mg) with 100 μg of the anti-DMRT1
monoclonal antibody 4F6 were mixed with 100 μL of EZveiw Red Protein G Affinity
Gel (Sigma), and incubated overnight at 4 ºC. Mouse normal IgG (Santa Cruz
Biotechnology; sc-2025) was used as a negative control. The gels were washed
twice with RIPA buffer, and the denatured proteins were separated by SDS-PAGE
(Perfect NT Gel W, 10–20% acrylamide, 28 wells; DRC Co. Ltd.). Silver staining
was performed with the 2D-SILVER STSIN II kit (Cosmo Bio 423413).