Rhodamine and fluorescein isothiocyanate conjugated secondary antibodies

GMCs were grown on coverslips in 6-well plates. After overnight adherence, cells were incubated with 30 mmol/L high glucose or mannitol for 24 h as described above and then were fixed in 4% paraformaldehyde (Pierce Biotechnology, USA) and permeabilized in 0.25% Triton X-100 (Sigma, USA). Cells were blocked in 5% goat serum, followed by incubation with anti-PIASy and anti-SUMO1 or anti-SUMO2/3 antibody (dilution 1 : 100) overnight at 4°C. After washing, cells were incubated with rhodamine- and fluorescein isothiocyanate-conjugated secondary antibodies (Bio-Synthesis) for 45 min in the dark. 4′,6′-Diamino-2-phenylindole (DAPI) was used to stain the nucleus in the cells. The coverslips were washed and imaged with a DMIRE2 laser scanning confocal microscope (Leica, Germany). The values of semiquantitative analysis for average intensity were assessed by Image-Pro Plus 6.0 software.

Mesangial cells were grown on coverslips in 6-well plates. Cells were fixed in 4% paraformaldehyde (Pierce) and permeabilized in 0.25% Triton X-100 (Sigma) for 10 min. Cells were washed twice in PBS and blocked in 5% goat serum for 1 h at room temperature, followed by incubation with anti-Smad4 and anti-SUMO2/3 antibodies overnight at 4°C. After washing, cells were incubated with rhodamine and fluorescein isothiocyanate-conjugated secondary antibodies (Bio-Synthesis) for 45 min in the dark. The coverslips were washed and mounted onto slides using mounting medium (Beyotime) and imaged with a DMIRE2 laser scanning confocal microscope (Leica, Germany).