Ab108421

Protein A magnetic beads (100 μL; Bio-Rad Biotechnology) were incubated with 5 μg DcR2 (ab108421; Abcam), Flag (ab205606; Abcam), and rabbit monoclonal IgG antibody (ab172730; Abcam) at room temperature for 1 h. Protein G magnetic beads (100 μL) were incubated with 5 μg GRP78 (ab212054; Abcam), Myc (ab32; Abcam), and mouse IgG antibody (ab131368; Abcam) at room temperature for 1 h. The above beads were washed and collected. In addition, the renal tissues and cells were lysed with ice-cold IP Lysis buffer on ice for 30 min and 10 min, respectively. The lysate was then mixed with the Protein A or G magnetic beads and incubated at 4 °C overnight to form the immune complex, following which 100 µL of SDS-PAGE loading buffer was added prior to incubation at 100 °C for 10 min. Finally, the samples were subjected to western blotting with anti-GRP78, anti-DcR2, anti-Myc, or anti-Flag antibody.

Western blot analysis was performed on renal lysates or cell extracts using the following primary antibodies: anti-DcR2 (ab108421; Abcam), anti-FLIP (ab8421; Abcam), anti-cleaved caspase 3 (ab214430; Abcam), anti-caspase 8 (ab25901; Abcam), caspase 3 (ab184787; Abcam), anti-caspase 7 (ab255818; Abcam), anti-cleaved caspase 7 (ab256469; Abcam), Akt (sc5298, Santa Cruz), pAkt (sc135650, Santa Cruz), and anti-GAPDH (BM3876; Boster). The intensity of each band was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).

Masson trichrome staining was performed in accordance with a standard protocol and analyzed under an Olympus microscope (Tokyo, Japan). All samples were incubated with primary anti-DcR2 (ab108421; Abcam, Cambridge, UK), anti-α-SMA (BM0002; Boster Biotechnology, Wuhan, China), anti-collagen IV (ab111742; Abcam), and anti-GRP78 (ab21685; Abcam). At least ten fields were randomly selected to evaluate the percentage of positive RTEC staining.