Fig. 2

HLA-B27 interacts with ALK2 and ALK5 in mLN lymphocytes from B27 rats. PLA was performed on lymphocytes isolated from (A-C) B7 or (D-F) B27 rats mLN, using w6.32 pan MHC-I Ab and anti-ALK2, -ALK3 or -ALK5. Images show representative field for each condition. Cells were stained for nuclei (Blue, DAPI) and PLA signal dots (RedHot, PLA kit probes). Scale bar: 10 μm. Images were treated with Fiji software. An unspecific PLA signal was homogeneously observed in all conditions. (I) Average number of PLA staining dots/cell in 2 to 4 independent experiments, showing a greater interaction between ALK2 or ALK5 and HLA-B27 than HLA-B7, whereas interaction was similar for ALK3 with both alleles. The background signal using single Abs was similar to the signal observed by combining anti-ALK Abs with anti-CD45RC (Supplementary Fig. 1). In conditions combining two Abs, approximately 300 to 400 cells per experiment were manually counted, whereas 150 to 250 cells per experiment were manually counted in single Ab conditions. Two-way ANOVA (factors: Ab and rat strain) followed by Bonferroni’s multiple-comparisons test was performed (*: p < 0.05 ***: p < 0.001). Vertical error bars show SEM