Ab51177

For western blotting, proteins were extracted from tissues or cultured cells using RIPA buffer containing phenylmethanesulfonylfluoride (PMSF) (Beyotime, Nantong, China). Equal amounts of protein (100 µg) were separated by SDS-PAGE on a 7.5%/12.5% gel and transferred to a PVDF membrane. Primary polyclonal antibodies targeting IκBα(ab7217), IκBα s36 (ab133462), IL-6 (ab6672), TGF-β (ab2486), PAI-1 (ab142349), SMAD3 (ab40584) and p-SMAD3 s425 (ab51177) were purchased from Abcam. The secondary antibodies used were HRP-conjugated anti-rabbit or anti-mouse antibodies and were purchased from Santa Cruz Biotechnology. The blots were developed using ECL reagent (Millipore, MA, USA). An equal amount of loaded protein in each lane was confirmed using a β-actin antibody. ImageJ software was used to quantify the integrated density of the band. ELISA Levels of PAI-1, TGF-β, CCL-17, IL-6 and CCL-22 were measured in the supernatants from cell cultures with a commerical ELISA kit (Thermo Fisher; BMS2033, 88-8350-86, EHCCL17, BMS213-2 and EMCCL22). The experiments were carried out according to the manufacturer's instructions.
3D co-culture system Differentiated THP-1 and 95D cells were cultured in a 3D Petri Dish (Micro-Tissues) (RI, USA) per manufacturer's instructions. Cell culture supernatants were collected daily for seven days. The cells were then collected, washed with PBS, and stained.