Lysis buffer

Dental pulp tissue was obtained by using a previously described method and was examined at the gene and protein level (Martin et al. 2002 (link)). Briefly, the pulp tissue was collected from extracted teeth with a number 330 bur at high speed with a water spray and then placed on ice and stored at −70 °C. HDPFs at the fourth passage were isolated as described above and were dissolved in RNA plus (Takara, Otsu, Japan) for qRT-PCR analysis or in lysis buffer (Santa Cruz Biotechnology, Calif., USA) for Western blotting and enzyme-linked immunosorbent assay (ELISA) analysis.

The cells were collected and suspended in lysis buffer (Beyotime Biotechnology, China). The entire lysate was incubated with antibodies against HuR (Santa Cruz Biotechnology, Cat#sc-5261), β-TrCP (Cell Signaling Technology, Cat#4394), or IgG (Beyotime Biotechnology, Cat#A7031). RNA-protein complexes were recovered with protein A/G plus-agarose (Santa Cruz Biotechnology, Cat#sc-2003) and then washed with lysis buffer four times. The precipited RNA was analyzed by qRT-PCR.

TJ protein levels were tested using semiquantitative immunoblotting. HK-2 cells were plated onto 100 mm-Petri dishes at 0.5 ~ 1 × 10⁵ cells/mL and cultured for siRNA transfection. Cells were harvested and lysed in lysis buffer (Santacruz, Heidelberg, Germany) containing protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice. Cell lysates were centrifuged at 14,000 x g for 20 min at 4°C and protein concentrations measured using Bradford protein assay kits (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were electrophoresed on SDS-polyacrylamide gels, transferred onto nitrocellulose membranes and blocked in 5% skim milk in PBST (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h. Membranes were probed overnight at 4°C with primary antibodies: rabbit polyclonal anti-occludin, rabbit polyclonal anti-ZO-1 or mouse monoclonal anti-claudin-2 (Zymed Labs, Jerusalem, Israel). Secondary antibodies were goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Sites of antibody-antigen reaction were viewed using enhanced chemiluminescence (GenDEPOT, Barker, TX), and band densities were quantified by densitometry using a laser scanner and Quantity One Software (Basic version 4.6.9, Bio-Rad, Hercules, CA).

The cortical tissue (30 mg) surrounding the lesioned areas were lysed in lysis buffer (Santa Cruz Biotechnology), and the total protein levels were quantified. A total of 25 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane that were blocked in 10% skimmed milk at room temperature for 2 h. The solution was incubated at 4°C overnight with primary antibodies against H3, H4, acetyl-H3, acetyl-H4, acetyl-Nrf2 (Cell Signaling Technology), cleaved caspase-3, LC3, Beclin, ATG-3 and ATG-7 (Cell Signaling Technology), Bax, ROS and GFAP (Abcam), Iba-1, Nrf2, HO-1, NQO1 and UGT1A1 (Santa Cruz Biotechnology Inc.), followed by incubation with appropriate secondary antibodies. Immunoblots were visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).

First, the whole proteins were extracted using RIP (radioimmunoprecipitation) lysis buffer (Santa Cruz Biotechnology, Inc). Second, using a semi‐dry blotting system, extracted proteins were loaded on an SDS gel and transferred to PVDF membranes. Then, incubation of the membrane with 0.5% Tween‐20 in PBS and 3% BSA was done for 2 hours at RT (room temperature). The next step was the incubation of the membrane with goat monoclonal antibodies for 2 hours. Further, HRP‐conjugated rabbit or mouse anti‐goat secondary antibodies were used for 1 hour at RT. All antibodies were purchased from Santa Cruz Biotechnology, Inc Finally, the ECL (enhanced chemiluminescence kit) was used to visualize the bands by Western blot imaging instrument (Sabz Co.). ImageJ software was used to qualify the density of bands and normalized to the density of the β‐actin band.