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. 2013 Oct 11;8(10):e75801. doi: 10.1371/journal.pone.0075801

Figure 5. The CTA1 and TCRα release activities do not co-fractionate with established ERAD cytosolic ATPases.

Figure 5

(A) P1 from CT-intoxicated cells was incubated with buffer, CE, or CE heat-inactivated (h.i.) at 65 or 95 degrees. The resulting S2′ were analyzed by immunoblotting with the indicated antibodies. (B) 1 ml CE was subjected to size exclusion chromatography by passage through a Bio-Sil SEC 250 column. The indicated even fractions were collected and used to generate S2′ from CT-intoxicated P1. Fractions were also directly analyzed for protein content by immunoblotting with the indicated antibodies (bottom panels). (C) As in A, except P1 derived from epoxomicin-treated cells expressing TCRα-HA was used. (D) 1 ml CE was subjected to size exclusion chromatography by passage through a Bio-Sil SEC 250 column. The indicated odd fractions were collected and used to generate S2′ from P1 derived from epoxomicin-treated cells expressing TCRα-HA. S2′ were treated with PNGase F prior to SDS-PAGE. Fractions were also directly analyzed for protein content by immunoblotting with the indicated antibodies (bottom panels).