Polyubiquitin

Eight of the nine putative cotton reference genes evaluated in this work, GhACT4 (actin gene family), GhEF1α5 (elongation factor 1-alpha), GhFBX6 (F-box family protein), GhPP2A1 (catalytic subunit of protein phosphatase 2A), GhMZA (clathrin adaptor complexes medium subunit family protein), GhPTB (polypyrimidine tract-binding protein homolog), GhGAPC2 (glyceraldehyde-3-phosphate dehydrogenase C-2), GhβTUB3 (β-tubulin), were selected according to their similarity to reference genes identified in Arabidopsis (Table 1) [6 (link)]. The sequences of possible G. hirsutum homologues were identified through a BLASTN against the database of the Green plant GB TAIR (The A. thaliana Information Resource, http://www.arabidopsis.org/). Only sequences that showed similarity higher than 1e-75 (E-value) were considered as putative homologous to the Arabidopsis genes and were selected for primer design. We also selected the gene encoding the poly-ubiquitin, GhUBQ14, commonly used in cotton for experiments of Northern blots and RT-qPCRs [26 (link),27 (link)] (Table 1). Primers were designed with Primer 3 software [28 (link)] using as criterion amplified products from 80 to 180 bp with a Tm of 60 ± 1°C (primer sequences are shown in Table 1). Both candidate reference and MADS-box genes were amplified from cDNA. Melting curve and gel electrophoresis analysis of the amplification products confirmed that the primers amplified only a single product with expected size (data not shown). Primer sets efficiencies were estimated for each experimental set by Miner software [29 (link)], and the values were used in all subsequent analysis (Table 2 and Additional file 2). Miner software pinpoints the starting and ending points of PCR exponential phase from raw fluorescence data, and estimates primer set amplification efficiencies through a nonlinear regression algorithm without the need of a standard curve.
Polymerase chain reactions were carried out in an optical 96-well plate with a Chromo4 Real time PCR Detector (BioRad) sequence detection system, using SYBR^®Green to monitor dsDNA synthesis. Reaction mixtures contained 10 μL of diluted cDNA (1:50), 0.2 μM of each primer, 50 μM of each dNTP, 1× PCR Buffer (Invitrogen), 3 mM MgCl2, 2 μL of SYBR^®Green I (Molecular Probes) water diluted (1:10000), and 0.25 units of Platinum Taq DNA polymerase (Invitrogen), in a total volume of 20 μL. Reaction mixtures were incubated for five minutes at 94°C, followed by 40 amplification cycles of 15 s at 94°C, 10 s at 60°C and 15 s at 72°C. PCR efficiencies and optimal quantification cycle threshold (Cq values were estimated using the online Real time PCR Miner tool [29 (link)]. For all reference and MADS-box genes studied, two independent biological samples of each experimental condition were evaluated in technical triplicates.

The firefly luciferase (FFLuc) and Renilla luciferase (Rluc) expression plasmids pIZ-Fluc and pAcIE1-Rluc have been previously described (41 (link)). The backbone used to generate the remainder of the expression constructs was a plasmid expressing FFLuc under control of the polyubiquitin promoter (PUb) (30 (link)), available from Addgene (catalog number 52891). The FFLuc coding sequence was removed, and the resulting plasmid was thereafter named pPUb. Gene sequences were obtained by extracting total RNA from Aag2 cells and synthesizing cDNA. Gene-specific primers were used to amplify the target gene of interest. The zeocin resistance gene was amplified by PCR from pIB/V5-His (Life Technologies, Inc.). A double-2A element from Thosea asigna virus was synthesized at Geneart, and tag sequences (myc or V5 tag) were added by PCR. All PCRs were carried out with KOD polymerase (Merck Millipore), and sequences of finalized constructs were verified via Sanger sequencing. The sequences are available upon request.

RNA was extracted from different organs and different fruit stages of blueberry plants, and Arabidopsis leaves using TaKaRa MiniBEST Plant RNA Extration Kit (TaKaRa, Shiga, Japan). qRT-PCR was performed with VcACTIN as a reference gene. Arabidopsis polyubiquitin 10 (AtUBQ10) was used as a reference gene. Single-stranded cDNA was obtained using a reverse transcription kit (TaKaRa, Shiga, Japan). 4 μg RNA was used as a template for cDNA synthesis. The procedure for cDNA synthesis was to incubate the reaction solution at 65°C for five minutes, 42°C for 60 minutes, and 70°C for 5 minutes. qPCR assays were performed on a CFX Connect real-time PCR assay system (Bio-Rad, USA) using the SYBR Green I chimeric fluorescence assay. The amplification reaction procedure was as follows: Stage 1 pre-denaturation, the mixture was reacted at 95°C for 30 sec, one cycle; Stage 2 cyclic reaction, 95°C for 10 sec, 60°C for 30 sec, 40 cycles; Stage 3 melting curve, 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec, one cycle. The qPCR results were all analyzed using the comparative CT method. Three biological replicates and three technical replicates were conducted. All primers used in this study are listed in Supplementary Table S1.

In total, 5 × 10⁶ CRC cells were harvested with a cell scraper, and RIPA lysis buffer (P0013B, Beyotime) was used to extract total protein. The concentration of total protein was measured with a bicinchoninic (BCA) protein assay kit (PC0020, Solarbio). Samples were mixed with 5x loading buffer and then heated at 100 °C for 10 min. Then, 25 μg of total protein was loaded onto SDS–PAGE gel, and a standard western blot procedure was performed. The separated proteins were transferred to PVDF membranes (IPVH00010, Millipore) and blocked with 5% nonfat milk. The membrane was incubated with primary antibodies overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The HRP-labeled proteins were visualized with an ECL kit (WBKLS0010, Millipore) and an automatic chemiluminescence image analysis system (5200 Multi, Tanon). All full-length uncropped western blots are provided in the Supplementary Material. The following antibodies were used: an anti-p-STAT3 antibody (Y705) (Cy6566, Abways), an anti-STAT3 antibody (sc-482, Santa Cruz), an anti-HuR rabbit polyclonal antibody (11910-1-AP, Proteintech), an anti-HuR mouse monoclonal antibody (mAb; 66549-1-Ig, Proteintech), an anti-β-actin antibody (30101ES60, Yeasen Biotech Co.), an anti-BCL2 antibody (12789-1-AP, Proteintech), an anti-C-MYC antibody (10828-1-AP, Proteintech), an anti-cyclin D1 antibody (sc-753, Santa Cruz), an anti-PARP antibody (#9542, CST), an anti-cleaved caspase-3 antibody (#9661, CST), an anti-caspase-3 antibody (#9662, CST), an anti-Flag antibody (F1804, Sigma), an anti-Ub (P4D1) mouse mAb (#3936, CST), an anti-K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (#8081, CST), an anti-β-TrCP antibody (sc-390629, Santa Cruz), anti-rabbit IgG (sc-2027, Santa Cruz), anti-mouse IgG (sc-2025, Santa Cruz), peroxidase-conjugated goat anti-rabbit IgG (33101ES60, Yeasen Biotech Co.), and peroxidase-conjugated goat anti-mouse IgG (33201ES60, Yeasen Biotech Co.).

The nine candidate genes that were selected for screening based on their role as reference genes in other plants were aligned with the garlic transcriptome established by our group, including ACT(Actin), EF1(elongationfactor1), UBC-E2(Ubiquitin-conjugating enzyme-E2), GAPDH(Glyceraldehyde-3-phosphatedehydrogenase), HIS3 (Histone H3), RPS5(ribosomalprotein S5), UBC(Ubiquitin-conjugating enzyme), UBQ(Polyubiquitin), 18S rRNA(18S ribosomal RNA). All RT-qPCR primers were designed with the Primer 5.0.