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. 2020 Apr 14;50(8):1209–1219. doi: 10.1002/eji.201948502

Figure 5.

Figure 5

Sera from AS patients contain IgG CD74‐autoantibodies that recognize accumulated CD74 degradation products in SPPL2a KO THP‐1 cells. (A, B) E.V. control and SPPL2a KO THP‐1 cells were stimulated with IFN‐γ for 24 h and lysed in homogenization buffer (HB) and subcellularly fractionated using a Percoll‐density gradient. Twelve fractions were collected with a volume of 500 μL (top fraction 1, bottom fraction 24). Fractions 1–7, 8–10, 11–12, 13–14, 15–16, 17–18, 19–20, 21, 22, 23, and 24 were collected and centrifuged to pellet the organelles and lysed in Laemmli buffer and analyzed for LAMP‐1, CD74 N‐terminus, and Na+/K+ ATPase expression using Western blot. (C) Protein‐G agarose beads were coated with sera from four AS patients and one HC. IFNy‐treated E.V. control and SPPL2a KO THP‐1 cells were lysed and proteins were immunoprecipitated using serum‐coated beads. Immunoprecipitates were examined using Western blot and analyzed for CD74 fragments. (A–C) Blots are representative of two independent experiments.