(a) Identification of the interaction of MANF and p65 by a yeast two-hybrid assay. (b) Co-localization of MANF and p65 in the primarily cultured FLS. The FLS isolated from normal rabbits were treated with LPS (10 μg/ml) for 48 hrs or TM (2.5 μg/ml) for 16 hrs, and then were stained with anti-MANF (red) and anti-p65 (green) antibodies. DAPI was used to stain the nuclei (blue). Scale bar = 10 µm. (c) Co-localization of MANF and p65 in the synovial tissue. The synovial tissues isolated from AIA rabbits were stained with anti-MANF (green) and anti-p65 (red) antibodies, respectively. DAPI was used to stain the nuclei (blue). Scale bar is 50 µm (upper panel) and 20 µm (lower panel), respectively. (d) Verifying the interaction of MANF and p65 by co-immunoprecipitation. The cells treated with TM (2.5 μg/ml) for 12 hrs were lysed for immunoprecipitation with an anti-p65 antibody, followed by Western blotting with the indicated antibodies. (e) MANF interacts with p65 in the nuclei. The cells were transfected with MANF-GFP, and then treated with or without TM (2.5 μg/ml) for 12 hrs. The cytosolic and nuclear fractions were immunoprecipitated with an anti-p65 antibody. Tubulin and histone H3 were used as the markers of the cytosol and nucleus, respectively. The experiments in all the panels were repeated for at least three times.