Hifiscript gdna removal rt mastermix kit

The qRT-PCR assays were performed to measure the expression levels of Snail, Cox-2, and Vegf-c. For 4T1, HuROE 4T1, and ΔHuR 4T1, the cells were treated with 1‰ DMSO or eltrombopag (5 μmol/L) for 12 h before RNA extraction. For the RAW264.7 cells, the model group was stimulated with LPS (100 ng/mL), and the eltrombopag groups were incubated with LPS (100 ng/mL) and eltrombopag (10 μmol/L or 20 μmol/L) for 12 h. The total cellular RNA was extracted using TRIzon Reagent (CWBIO, Nanjing, China) following the manufacturer’s instructions, and the RNA concentration was quantified with Nanodrop (DeNovix, USA). cDNAs were conducted using the HiFiScript gDNA Removal RT MasterMix kit (CWBIO, Nanjing, China), and quantitative PCR was performed with the ABI QuantStudio 6 Flex system (Thermo Fisher Scientific, USA) using MagicSYBR green master mix kit (CWBIO, Nanjing, China). The mRNA levels of selected genes were normalized to β-actin and analyzed with 2^−ΔΔCt method. The primers synthesized by Sangon Biotech (Shanghai, China) were as follows: Snail (F: 5′-CACAC GCTGC CTTGT GTCT3′ and R: 5′-GGTCA GCAAA AGCAC GGTT-3′), Cox-2 (F: 5′-GGTGC CTGGT CTGAT GATGT ATGC-3′ and R: 5′-GGATG CTCCT GCTTG AGTAT GTCG-3′), Vegf-c (F: 5′-CTACA GATGT GGGGG TTGCT-3′ and R: 5′-GATTG GCAAA ACTGA TTGTG AC-3′), and β-actin (F: 5′-GTGGG CCGCT CTAGG CACCA A-3′ and R: 5′-TGGCT TTAGG GTTCA GGGGG-3′).

RIP assays were carried out as described with minor modifications [49 (link)]. The 4T1 cells and RAW264.7 cells were treated with eltrombopag as described in Section 4.7. Cell lysates were collected with RIPA Lysis Buffer (Sangong Biotech, Shanghai, China), and protein quantification was measured using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The experimental flow is shown in Figure 4D. Equal amounts of cell lysates (300 μg) were pre-bound with protein A/G beads (SC-2003, Santa Cruz Biotechnology, Dallas, TX, USA), and then HuR antibody (1:50, 12,582 s, Cell Signaling Technology, USA) was added to immunoprecipitation HuR-mRNAs complexes for 12 h at 4 °C. The mRNAs were isolated using TRIzon Reagent (CWBIO, Nanjing, China), and the cDNA was synthesized with the HiFiScript gDNA Removal RT MasterMix kit (CWBIO, Nanjing, China). The HuR-binding mRNAs (Snail, Cox-2, and Vegf-c) and non-HuR-binding mRNA (Gapdh) were detected with qRT-PCR and analyzed by the 2^−ΔΔCt method. Mouse IgG (Sepharose^® Bead Conjugate, 3420 S, Cell Signaling Technology) was used as a negative control. The primers used in the RIP analysis are listed in Section 4.7. The primers of Gapdh were 5′-AGGTC GGTGT GAACG GATTT G-3′ (F) and 5′-GGGGT CGTTG ATGGC AACA-3′ (R).