M mlv reverse transcriptase m1701

Total RNA was isolated from HeLa cells and reverse-transcribed using, respectively, QIAzol Lysis Reagent (Qiagen, Milan, Italy) and MMLV Reverse Transcriptase M1701 (Promega, Milan, Italy), as previously described [58 (link)]. In Table 1 are listed the primers used for AQP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 amplification. Briefly, QuantiFast SYBRGreen PCR Master Mix (Qiagen, Milan, Italy) was used to perform the qPCR. Thermal cycle conditions for qPCR as well as the procedures used were previously reported [36 (link)]. Melt curve analysis and separation of the PCR products by gel agarose electrophoresis were performed to verify the presence of single and specific products [36 (link)]. Relative mRNA quantitation was expressed as ΔCt, obtained by subtracting the Ct(housekeeping gene) from Ct(AQP gene). Note that high ΔCt values reflect low mRNA expression levels.

To generate Arabidopsis FPTP1 over-expression transgenic plants, the coding sequence of FPTP1 were amplified and inserted into the modified pCAMBIA1300-Super vector under control of 35S promoter. These vectors were transformed into Agrobacterium strain GV3101 and subsequently introduced into the Arabidopsis wile-type plants.
With the aim of identifying the contribution of PTPs in ABA accumulation, RNA was extracted using an RNeasy Plant Minikit (QIAGEN), and cDNA was synthesized using M-MLV Reverse Transcriptase (M1701) (Promega). 1 μg RNA was mixed thoroughly with 1 μg Oligo (dT)₁₅, and treated at 70°C for 5 min. Subsequently, the following were added: 5 μL M-MLV buffer, 1.25 μL dNTP Mix, 1 μL M-MLV, and 0.6 μL RNase inhibitor. The resulting 25 μL reaction was incubated for 1 h at 37°C. PCR primers were showed in Additional file 1. PCR cycles included 94°C 5 min, (94°C 45 s, 58°C 45 s, 72°C 1 min) 35 cycles, 72°C 7 min.

Antibodies against Iκκβ (ab32135) and p65 (ab7970) and Alexa Fluor 555 Donkey anti-Rb IgG (H + L) antibody (ab150074) were obtained from Abcam (Cambridge, MA, USA). The other antibodies used were as follows: anti-IKB-α (sc-1643) and anti-p-IKB-α (sc-8404) (Santa Cruz Biotechnology, CA, USA), anti-p-p65 (3033S; Cell Signaling Technology, Danvers, MA, USA), and anti-NFKB1 (NF-κB p50) (14220-1-AP; ProteinTech, Rosemont, IL, USA). Anti-GAPDH (H301; TransGen Biotech, Beijing, China) was used as normalized control. Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (CSB-E04638h), IL-8 (CSB-e04641h), and TNF-α (CSB-EQ023955HU) were obtained from Cusabio Biotech (Wuhan, China). M-MLV Reverse Transcriptase (M1701) and GoTaq qPCR Master Mix (A6001) were obtained from Promega (Madison, WI, USA).