Anti usf1 antibody

To clarify whether USF1 bound to the promoters of tight junction related proteins, ChIP assay was performed. Simple ChIP Enzymatic Chromatin IP Kit (Cell signaling Technology, Danvers, Massachusetts, USA) was used according to the manufacturer's instruction. In brief, cells were crosslinked with 1% formaldehyde for 10 min and treated with glycine for 5 min. Cells were collected in cold lysis buffer containing PMSF. Chromatin was digested by Micrococcal Nuclease for 20 min at 37°C. Before adding antibody 2% input reference was removed and stored at −20°C. Three micrograms of anti-USF1 antibody (Santa Cruz Biotechnology, CA, USA) was added in the immunoprecipitation sample with gentle shaking at 4°C overnight. Negative control was conducted with normal rabbit IgG. The chromatinimmune complex was eluted from the antibody/Protein G beads with gentle vortexing at 65°C for 30 min.
The DNA crosslinks was reversed by 5 mol/L NaCl and Proteinase K at 65°C for 2 h and DNA was purified. PCR was performed to amplify immunoprecipitated DNA. The detail informations of specific primers were listed in Table 1.

To induce the development of DNA‐protein cross‐linking, Huh7 cells were fixed with formaldehyde for 10 minutes. Then, we used a sonicator for separation of the chromatin. The harvested cells were split into two containers that were separately incubated with an anti‐USF1 antibody (Santa‐Cruz Biotechnology) and NC rabbit anti‐IgG antibody (Abcam) at 4°C overnight. After centrifugation for 10 minutes at 12 000 g at 4°C, protein sepharose/agarose was used to precipitate the protein‐DNA conjugates which were centrifuged again at 12 000 g for 5 minutes, and the supernatant discarded. The heterogenous conjugates were isolated, and cross‐linkages were reversed by incubation overnight at 65°C. Chloroform/phenol purification of the harvested DNA prepared samples for PCR. One set of primers ensured amplification of the ROMO1 promoter region binding USF1 (site 1). An additional set of primers ensured amplification of the DNA sequence beyond the ROMO1 promoter DNA and served as a negative control. Both sets of primers together were used for qRT‐PCR to confirm the ROMO1 DNA sequence binding USF1. A chromatin immunoprecipitation (ChIP) assay was carried out with the purified DNA fragment following lncRNA TUG1 silencing. In other control experiments, sh‐NC was used.

The protein levels of DNMT1, TGFb1, USF1 and SREBP1 were analyzed by immunoblotting using anti-DNMT1 antibody (Abcam, no. ab188453), anti-TGFb1 antibody (Abcam, no. ab92486), anti-USF1 antibody (Santa Cruz Biotechnology, no. sc-229) and anti-SREBP1 antibody ((R&D Systems, no. NB100-60545SS)). Expression levels of beta-actin were used for normalization. The protein level of beta-actin was analyzed using anti-beta-actin antibody (Abcam, no. ab8227). Wide-view prestained protein size marker III was used as size marker of DNMT1 protein (Wako, no. 230-02461).