Ab92486

RNA was extracted from cultured cells by using TRIzol. For serum samples, RNA was isolated by using TRIzol LS from 200 μl serum. Cel‐miR‐39 was added at 2 nM as a spike‐in control. Relative mRNA or miRNA expression was determined by using SYBR Green (Bio‐Rad) or TaqMan probe‐participated qPCRs, GAPDH and U6 served as internal controls for normalization of mRNA and miRNA expression, respectively. The sequences for qPCR primers are listed in Appendix Table S2. Proteins were isolated by using RIPA lysis buffer and separated by SDS–PAGE. After being transferred to PVDF membranes, membranes were incubated with various antibodies as indicated. Protein bands were detected by electrochemiluminescence with horseradish peroxidase‐labeled secondary antibodies. The used primary antibodies were as follows: TGF‐β (Abcam, ab92486, 1:1,000); TGFBR2 (Santa Cruz, sc‐17792, 1:500); β‐catenin (Abcam, ab32572, 1:1,000); CTGF (Santa Cruz, sc‐365970, 1:1,000); IL‐1β (Abcam, ab2105, 1:1,000); and ET‐1 (Abcam, ab2786, 1:1,000). Secondary antibodies used were as follows: anti‐mouse (Jackson, 515‐035‐003, 1:5,000) and anti‐rabbit (Jackson, 111‐035‐045, 1:5,000).

pTDM was shaped into a hollow circular column (3 mm × 2 mm × 4 mm) for subcutaneous implant after ethylene oxide disinfection. Shanghai Model Organisms Center, Inc. was entrusted to establish an animal disease model of B6;129S-Sod1^tm1LebJ (SOD1^−/−) of C57 mice. After anesthetized with isoflurane, the prepared xenogeneic bioroots with or without RSG pretreated were implanted subcutaneously on the back of 4-month-old SOD1^−/− C57 mice. The mice were killed 2 months after implantation and the samples were fixed at 4°C in 4% PFA overnight, and then demineralized with 10% EDTA for 4 months. Paraffin sections were prepared for subsequent histological staining as previously described.^{2 (link)} Anti-TGFβ1 (abcam, ab92486), anti-Periostin (H-300) (santa cruz, sc-67233) and anti-MMP2 (abcam, ab92536) primary antibodies were applied at a dilution of 1:200.

Total protein was isolated from flash-frozen liver tissue using RIPA buffer (IL6, TGFB) and run on a 2–10% gradient gel and transferred to a PVDF membrane. For collagen quantification protein lysates were isolated from frozen liver tissue using 0.075M citrate buffer (pH 6) and resolved using 2–8% gradient gel and transferred to PVDF under non-denaturing conditions. Membranes were blocked with 4% BSA and probed with antibodies against TGFB (Abcam, ab92486) IL6 (Santa Cruz Biotechnology: (m-19), sc-1265) HSP90 (Cell signaling, #4874) or COL1A1 (Abcam, ab34710). Protein expression was quantified and normalized to HSP90 using ImageJ. Latent TGFB-L (44kDa) and mature TGFB-M (13kDA) were detected using the same antibody (ab92486). For fold-expression of proteins expression from ethanol fed from mice was normalized to pair-fed controls.