Irdye 800cw conjugated goat polyclonal anti mouse igg

Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA). PKC inhibitor Gö6983, PI3K inhibitor LY294202, and MEK1/2 inhibitor U0126 were purchased from Calbiochem (San Diego, CA, USA). AT1 receptor blocker candesartan was from AstraZeneca. Neutralizing anti-IGFR1 antibody, MAB 391, was bought from R & D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium/F12 (DMEM/F12) was from Life Technologies (Carlsbad, CA, USA). Nu serumTM and ITS+premix were obtained from BD Biosciences (Bedford, MA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), anti-ERK1/2, and anti-AKT antibodies were from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies [IRDye 800CW Conjugated Goat (polyclonal) anti-mouse IgG and IRDye 680 Conjugated Goat (polyclonal) anti-rabbit IgG] were products of LI-COR Biosciences (Lincoln, NE, USA). High-capacity cDNA reverse transcription kit was from Applied Biosystems. LightCycler® 480 SYBR Green I Master was purchased from Roche Applied Science. Trizol reagent and all other reagents came from Sigma-Aldrich (St. Louis, MO, USA).

Total RNA was extracted from N. benthamiana leaves infiltrated with A. tumefaciens, non-transgenic and transgenic soybean leaves, respectively, using the Trizol reagent (Cat no. 15596-026, Invitrogen, Carlsbad, CA, United States) following the instructions of the manufacturer. The RNA specimens were electrophoresed on 15% polyacrylamide/7 M urea gels and transferred to Hybond N⁺ membranes (Amersham) using a semidry blotter (Bio-rad, Hercules, CA, United States). The membranes were UV cross-linked in a HL-2000 HybriLinker™ (AnalytikJena: UVP) at 120,000 μJ/cm² energy for 4 min and separately hybridized with five different digoxin-UTP-labeled RNA probes. The mouse anti-digoxin monoclonal antibody (1:10000, Jackson ImmunoResearch) and IRDye 800CW-conjugated goat (polyclonal) anti-mouse IgG (1:15000; H + L; LI-COR Biosciences) secondary antibodies were used to evaluate siRNA production. The membranes were visualized using an LICOR Odyssey scanner with excitation at 700 and 800 nm. For Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay, the 320 bp SMV CP and 1,812 bp GUS bands were amplified from pSMV-GUS-infiltrated N. benthamiana leaves. The Western blot analyses were performed according to previously described protocols and the size of CP protein is ∼ 30 kDa (Jiang et al., 2019 (link)).

The genomic DNA was extracted from soybean hairy roots by cetyltrimethylammonium bromide method with minor modifications [43 (link)]. Insertion of the P19 and CP gene into soybean genome was confirmed by PCR using the genomic DNA of hairy root transformants, respectively. Forward primer 5′-ATGGAACGAGCTATACAAGGAA-3′ and reverse primer 5′-CTCGCTTTCTTTTTCGAAGGT-3′ were used to amplify the 516 bp P19 fragment. Forward primer 5′-GAAGGAGACATGGATGCAG-3′ and reverse primer 5′-CTTGCAGTGTGCCTTTCAG-3′ were used to amplify the 731 bp CP fragment. The GFP fluorescence of hairy roots transformed with the vector control, P19 or CP overexpression vector were monitored under stereo fluorescence microscope (OLYMPUS MVX10, Japan). The proteins of GFP-positive transgenic hairy roots were extracted and the protein concentrations were determined by the Bradford assay procedure using bovine serum albumin as the standard [44 (link)]: 10 μg protein samples were separated with 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Immobilon). Anti-GFP monoclonal antibody (1:3000; Sigma-Aldrich) and IRDye 800CW-conjugated goat (polyclonal) anti-mouse IgG (1:10,000; H + L; LI-COR Biosciences) secondary antibodies were used to further evaluate the gene expression in soybean hairy roots. The membranes were visualized using a LI-COR Odyssey scanner with excitation at 700 and 800 nm.