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. 2015 Sep 23;29(12):5081–5089. doi: 10.1096/fj.14-269795

Figure 4.

Figure 4.

Identification and verification of the K8−/− autoantigens. A, B) Total mouse liver lysates were separated by isoelectric focusing then SDS-PAGE. Proteins were stained by Coomassie Brilliant Blue (A) or blotted with K8−/− serum that recognizes all 4 autoantigens. Liver lysates separated only by SDS-PAGE are included (1D, right side of the panels). C) GDH1 (lanes 1) and total liver lysates (lanes 2) were separated by SDS-PAGE on the same gels for each serum, transferred, and analyzed by immunoblotting using K8−/− sera containing the C, B, or C and B autoantibodies, or with K8+/+ serum as control. GDH1 is recognized by K8−/− sera containing antibody B but not C (indicated by boxes). D) Purified GDH1, liver lysates, and NIH-3T3 lysates were separated on parallel gels and transferred for blotting. Prior to blotting, the K8−/− serum was preabsorbed (right panel) or not (left panel) with GDH1, then used for immunoblotting. E) Purified HMGCS2 was immunoblotted with K8−/− serum (lane 1) or K8+/+ serum (lane 2). F) HMGCS2 was immunoblotted with 2 independent K8−/− sera (serum #1 and serum #2) containing antibody C that had been preabsorbed (lanes 2) or not (lanes 1) with purified HMGCS2. Note that preabsorption leads to a marked reduction in autoantibody reactivity.