Hiscript q rt supermix for qpcr

Total RNA was extracted using the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, Norcross, GA, USA) and subsequently converted to cDNA with a reverse transcription kit HiScript Q RT SuperMix for qPCR (Vazyme, Biotech). qPCRs were performed with AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech) and the ABI 7500 Real-Time PCR amplifier (Applied Biosystems, Foster City, CA, USA). β-tubulin gene was used as the internal reference, and qPCR was used to analyze the specific mRNA expression level of the pcmfs gene. The qPCR amplification program was as follows: amplification at 95°C for 3 min, amplification at 95°C for 3 s, amplification at 60°C for 32 s, 40 cycles, and amplification at 72°C for 3 s. The relative gene expression was analyzed according to the 2^−∆∆CT method. Sequences of the primers used for qPCR are listed in Table S1.

Total RNA was extracted from cells or tissues using TRIzol reagent according to the manufacturer's instructions (Invitrogen, CA, USA). Reverse transcription (RT) was performed with HiScript Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd, Nanjing, China). RT‒PCR was performed in triplicate with a SYBR Green PCR Kit (Vazyme Biotech Co., Ltd, Nanjing, China) on an Applied Biosystems 7900HT sequence detection system (Applied Biosystems). The primers used are listed in Supplementary Table 2.

Total RNAs were isolated with TRIzol reagent (Invitrogen). Equal amounts of RNA (1 μg) from each sample were used for cDNA synthesis using HiScriptQ RT SuperMix for qPCR (Vazyme, Nanjing, China). qPCR was performed on the ABI StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using GoTaq qPCR Master Mix assay (Promega) and analyzed using StepOne Software v2.1 (Applied Biosystems). 2−ΔCT method was used to calculate gene expression levels. For sample loading control, GAPDH were tested. Primers used for qPCR amplification are available in Table S1.
Sample protein extraction and concentration determination of whole cells were performed as previously described [47 (link)]. Cytoplasmic and nuclear proteins were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions. Briefly, equal amounts of protein were run on SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5% non-fat dry milk and probed with antibodies. The following antibodies were used: GAPDH (KangChen Bio-tech, Shanghai, China), E-cadherin, N-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ, USA), Wnt5a (R&D systems, Minneapolis, MN, USA), Arf6, Vimentin (Abcam, New Terriories, HK, China), ERK, P-ERK, Histone H3, HA and GFP antibodies (Cell Signaling Technology, Danvers, MA, USA). Protein bands were detected by incubating with HRP-conjugated antibodies (Santa Cruz, CA, USA) and visualized with ECL reagent (Millipore, Billerica, MA, USA).