Stepone real time pcr machine

The total RNA of the transformants and the wild type was isolated from the mycelia of 2-day-old CM broth using a Qiagen RNeasy plant minikit (Qiagen, New Delhi, India) per the manufacturer’s instructions. Genomic DNA contamination was removed using RNase-free DNase (Qiagen). The integrity was checked on a 1% agarose gel, and the RNA was quantified using a Bio-Rad spectrophotometer. First-strand cDNA was synthesized using a Thermo Scientific RevertAid H minus first-strand cDNA synthesis kit. qRT-PCR was performed on an Applied Biosystems Step One real-time PCR machine using Power SYBR green PCR master mix (Applied Biosystems, Woolston, United Kingdom). The relative amounts of the target gene transcripts in terms of fold change were calculated using the expression 2^–ΔΔCT, where “CT” represents “threshold cycle” and “ΔΔCT” represents (CT_{gene of interest} – CT_actin)_{test condition} – (CT_{gene of interest} – CT_actin)_control. Each real‐time PCR was performed in triplicate, and values for each gene were normalized to the expression level of the wild‐type B157 isolate.

The protein stability and protein-ligand interactions were assessed by a thermal shift assay. In this assay, the quantum yield of the dye increases when it binds the exposed hydrophobic surfaces of the denatured proteins. Prior to the addition of 5× SYPRO Orange (Sigma-Aldrich), 2 μM EphB1 was incubated with dimethylsulfoxide or 10 μM dasatinib for 1 h. The proteins were heat denatured from 5 °C to 95 °C and fluorescence (excitation/emission 470 nm/570 nm) was recorded at 2 °C intervals in a StepOne Real-Time PCR machine (Applied Biosystems). The melt curves were normalized to obtain relative fluorescence values between 0 and 1. The normalized melt curves were fit to the Boltzmann sigmoidal equation in GraphPad Prism, version 9 (www.graphpad.com) to derive the T_m’s (V₅₀). The statistical significance was determined using a two-way ANOVA test in GraphPad Prism, version 9.

For the PAFR mRNA expression analysis, A549, H1299, KBM, and KBP cell lines were homogenized using an RLT buffer containing β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) in a bullet blender (Next Advance, Inc., Averill Park, NY, USA) and carbide beads. Total RNA was extracted from these cell lines using an RNaesy Plus mini kit as per the manual’s protocol, and quantified with Nanodrop 2000c (Thermo Scientific, Vernon Hills, IL, USA). For the analysis of miR-149-5p, cyclin D1, and FOXM1 mRNA expression, A549 cells were transfected with or without the miR-149-5p mimic or inhibitor and incubated for 6 h, followed by treatment with or without CPAF and incubated for 48 h. The control cells were transfected with Scr ctrl. Total RNA samples were extracted as described above. A High-Capacity cDNA Reverse Transcription kit containing random hexamers was used to transcribe RNA samples to cDNA as per the manufacturer’s instructions, and a SYBR green-based quantitative qPCR method was performed as per the manufacturer’s instructions and as previously reported [31 (link),39 (link)]. The fluorescence was detected using a StepOne Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). We used GAPDH, U6, and 18s as endogenous controls to normalize PAFR, miR-149, and cyclin D1 expression. The quantification of the PCR product was analyzed using the 2^−∆∆Ct method.

Total RNA (1 μg) was reverse transcribed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, United States). Quantitative reverse transcription PCR was performed with the SYBR master mix and StepOne real-time PCR machine (Applied Biosystems, Waltham, MA, United States). The thermal cycling program was as follows: 95°C for 20 s and 40 cycles of 95°C for 3 s and 60°C for 30 s. Relative fold change in gene expression normalized to Atef1a (eukaryotic translation elongation factor 1 alpha) or Bcactin (Bc1G_08198) was calculated by the comparative cycle threshold 2^–ΔΔCt method. Primers used in qRT-PCR analysis of A. thaliana are listed in Supplementary Table 6 and for B. cinerea in Supplementary Table 7.

Total RNA was extracted using miRNeasy kit (Cat. no. 217004; QIAGEN). cDNA synthesis was carried out using the RT2 first stand kit (QIAGEN) cDNA synthesis kit following the manufacturer’s protocol. qPCR then was carried out using Maxima SYBR Green Supermix (Life Technologies) in an Applied Biosystems StepOne Real-time PCR machine. 18S rRNA was used as the internal control for all samples. For human UBE2D2, the following primers were used: 5′-GGTCACAGTGGTCTCCAGCACTAA-3′ and 5′-ACTTCTGAGTCCATTCCCGAGCT-3′. For human UBE2L3, the following primers were used: 5′-CATCGATGAGAAGGGGCAG and 5′-ACTTGGTCAGTCTTGGTGGC-3′.

Total RNA was extracted by using RNeasy Mini Kit (Qiagen) and DNase I treated (Invitrogen). Briefly, 1 µg RNA was reverse‐transcribed using SuperScript III reverse transcriptase (Sigma‐Aldrich) following the manufacturer’s protocol. RT‐qPCR was designed following the MIQE guidelines (Bustin et al., 2009 (link)) with gene specific primers (Table S1). EF1α (accession no. TC19582 (At5g60390)) and PP2A (accession no. TC21939 (At1g13320)) (Liu et al., 2012 (link)) were used as reference genes in N. benthamiana and PEX4 (or UBC; accession no. AT5G25760) in Arabidopsis thaliana (Dekkers et al., 2012 (link)). Each 12.5 μl reaction contained 1× GoTaq® qPCR Master Mix, 1 μM of each primer, 1.4 mM magnesium chloride (MgCl₂), 2.4 μM CXR reference dye and a cDNA quantity of c. 25 ng. The PCR program was set on a StepOne™ Real‐Time PCR Machine (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) as follows: 95°C for 15 min followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. A melting curve was generated at the end of the PCR program and 2‐ΔΔCt value (Livak & Schmittgen, 2001 (link)) was calculated to determine the relative expression of NbSIZ1. Three technical replicates were performed in each run and three biological replicates were carried out.