Mgcl2 stock solution

Cinobufagin (CBG) was purchased from Pusi Biotechnology Co. Ltd, and the purity of CBG was 99.21%. For each experiment, CBG was freshly prepared by dissolving in DMSO (Sigma, USA). Bleomycin (BLM) was acquired from Nippon Kayaku (Tokyo, Japan). TRIzol reagent was from Ambion Life Technology. DEPC TREATED H₂O, RNASE AWAY H₂O and SYBR Green Real-time PCR Master Mix were from Life Technologies. M-MLV Reverse Transcriptase was purchased from Promega, and PCR Buffer without MgCl₂ and MgCl₂ Stock Solution were acquired from Roche. In addition, Recombinant Ribonuclease Inhibitor, dATP, dTTP, dCTP, and dGTP were acquired from Takara. RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology. The primary antibodies described in the study include: anti-E-cadherin, anti-Vimentin, anti-fibronectin, and anti-collagen I (Affinity Biosciences, OH, USA) and anti-GAPDH, Smad3, P-Smad3, ERK, P-ERK, JNK, P-JNK, P38, P-P38 antibody (Cell Signaling Technology, Boston, United States); α-SMA and β-catenin antibody were from Santa Cruz Biotechnology (China) and Protein-Tech (China), respectively. The secondary antibodies anti-rabbit IgG (H+L) and anti-mouse IgG (H+L) were from Applygen (Beijing). Polyethylenimine, Linear purchased from Tianjin Hao Trading. β-catenin plasmid was purchased from MiaoLingPlasmid. ELISA kits were purchased from Beijing Suobao Technology.

The conventional 16S rRNA PCR was performed as described by Pospischil et al. [35 (link)], using the primers 16S IGF (short) and 16S IGR (short) to amplify a sequence of 298 base pairs (bp) (Table 2).
Per sample, a 50 μl reaction mix was prepared, containing 1 μl (< 150 ng/μl) sample template, 1x PCR buffer with MgCl₂ (Roche Diagnostics GmbH), 0.5 mM MgCl₂ Stock Solution (Roche Diagnostics GmbH), 0.2 nM dNTP (PCR Nucleotide Mix, Roche Diagnostics GmbH), 300 nM of both primers and 0.02 U/μl FastStart Taq DNA Polymerase.
Cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 60 s, 65°C for 60 s and 72°C for 90 s and a final extension of 72°C for 10 min.