Anti v5 hrp antibody

The cDNAs encoding arylsulfataselike genes of P. xylostella (PxGSS1, PxGSS2, PxGSS3, and PxSulfD), Bombyx mori SulfC1 (BmC1), and Yponomeuta cagnagella C (YcC) were amplified by PCR using gene-specific primers (supplementary table S1, Supplementary Material online), which included a 5′ Kozak sequence and lacked the native stop codon to facilitate epitope and His-tag fusion expression. PCR products were ligated into a pIB/V5-His TOPO TA vector (Invitrogen), and correct sequence and orientation were verified by Sanger sequencing. Sf9 cells were cultivated in GIBCO Sf-900 II SFM (Invitrogen) on six-well plates at 27 °C until 70–90% confluence was achieved. Transfection was performed with FuGENE HD (Promega) following the manufacturer’s protocol. At 72 h after transfection, the culture medium of Sf9 cells was harvested and aliquots were directly used for blotting or for activity assays. Expressed proteins were detected with an anti-V5 HRP antibody (Thermo Fisher Scientific) using the SuperSignal West HisProbe Kit (Pierce).

FHIP1B KO and FHIP2A KO clones were transfected with TagRFP-T-V5 under a mutated CMV promoter (Ferreira et al., 2011 (link)) using a retroviral infection/MSCV-driven expression system as described previously (Sowa et al., 2009 (link)). Briefly, plasmid DNA (retroviral pMSCV with FHIP1B-TagRFPT-V5, FHIP2A-TagRFPT-V5 or TagRFPT-V5 genes inserted) along with viral helper constructs (retroviral MSCV-vsvg, MSCV-gag/pol) were diluted in OptiMEM (Gibco) and combined with 1 μg/μl polyethylenimine (PEI; Polysciences Inc) in a 3:1 ratio of PEI:DNA concentration. The transfection mixture was added to 293T cells, followed by incubation for 12–16 hr. Fresh DMEM was added to the cells, followed by a 24-hr incubation to allow virus production. Viral supernatant was collected, filtered, and added to recipient U2OS cells along with 1 μg/ml polybrene for infection. Stable cell lines were established by hygromycin selection (400 μg/ml) for 48–72 hr. Expression of ectopic proteins was confirmed via western blotting with FHIP1B and FHIP2A-specific antibodies as well as anti-V5-HRP antibody (Thermo Fisher Scientific, mouse monoclonal, Cat #: R96025). After recovery, stable cell lines were grown in DMEM containing 10% FBS, 1% PenStrep, and 200 μg/ml hygromycin.

Pol IV, Pol V, and Pol II were affinity-purified by virtue of their FLAG-tagged NRPD1, NRPE1, or NRPB2 subunits as described previously (26 (link)). To reconstitute Pol IV-RDR2 complexes, Pol IV expressed in a rdr2 null mutant background was immobilized on anti-FLAG M2 resin then incubated with 1 µM recombinant RDR2 for 30 min at 4 °C in 50 mM Hepes-KOH pH 7.5, 150 mM NaCl. The resin was then washed three times with 100 μL of the same buffer to remove unbound proteins then subjected to SDS-PAGE and immunoblotting using anti-FLAG-HRP (Sigma Aldrich) or antibodies recognizing native NRPD1, NRPE1, or NRPB2. Anti-V5-HRP antibody (Invitrogen) was used to identify recombinant RDR2.

Yeast DOB were obtained in powder form from MP Biomedicals (Solon, OH, USA). Cell culture plastics were from BD Falcon (San Jose, CA, USA). Dextrose, galactose, and raffinose were obtained from Sigma (St. Louis MO, USA). Glass beads for yeast cell lysis were from B. Braun Biotech (Allentown, PA, USA). Anti-V5-HRP antibody was from Invitrogen (Carlsbad, CA, USA). Mutagenesis reagents were obtained from Agilent (Santa Clara, CA, USA). All other chemicals were reagent grade or better, were purchased from Sigma (St. Louis MO, USA) and used without additional purification.

Cells were harvested 48 hours after transfection or treatment with TGF-β ligand and lysed in lysis buffer (20mM HEPES, 100mM KCl, 0.2mM EDTA, 0.5mM DTT, 2.5% Glycerol, and protease inhibitor (Roche)). 5–10 μg of total protein were loaded in each lane of 10% SDS PAGE gels and transferred to PVDF membranes. Anti-V5-HRP antibody (Invitrogen,CA, 46–0708) was utilized to detect and confirm V5-tagged vFLIP and vCyclin expression from plasmid constructs. Primary antibody for detecting SMAD2 was purchased from Cell Signaling Technology Inc. (Beverly, MA, Cat #3103).

We chose four exemplar species, one from each taxonomic family, for downstream analysis: A. asperrimus, P. schultei, R. artemis, and S. sipylus. We designed gene-specific forward and reverse primers (Table S1) to amplify the complete ORF of each putative enzyme from the cDNA, and cloned them into Top10 cells with the pIB/V5-His TOPO/TA^® vector (Invitrogen). We included Kozak sequences (RCCATGG) at the 3′ end of the forward primers and did not include the stop codon in the reverse primers. Colony PCR or direct sequencing was done to ensure the genes were cloned in the correct direction, then we extracted the plasmids with a GeneJET™ Plasmid Miniprep Kit (Thermo Scientific) and transfected them into Sf9 cells (Invitrogen) using the reagent FuGENE HD (Promega). Culture medium was harvested after 72 hours incubation at 27 °C and centrifuged, and the supernatant tested for successful expression via Western Blot (Figure S1) with anti-V5-HRP antibody (Invitrogen). Plate assays for substrate activity were performed on the individual enzymes following the same protocol as the whole gut extracts.