Figure 3.

Protease protection assay suggests a type II membrane topology.A, microsomes prepared by Dounce homogenization of HEK293 cells expressing either S1R-WT, S1R-BAP, or H2a-BAP were incubated as described in Experimental procedures, with or without proteinase K, before or after treatment with 1% SDS at 100 °C as indicated. Samples were then subjected to immunoblot with the antibodies indicated on the right. S1R and CNX were detected using antibodies recognizing epitopes toward the C-terminus. S1R-BAP and H2a-BAP were detected with antibodies against the SV5 tag at the C-terminus and the luminal control, BiP-RFP, was detected with anti-RFP. Microsomes treated with proteinase K without prior treatment with SDS protect the luminal and transmembrane regions of the proteins. For S1R, S1R-BAP, and H2a-BAP, a shift in migration due to cleavage by proteinase K of the cytosolic tails is indicated. Results are shown for a representative experiment of four independent repeat experiments. B, scheme of the results, which show type II membrane topology for S1R, S1R-BAP, and H2a-BAP (control for a type II membrane protein). The luminal control, BiP-RFP, was completely protected in the absence of SDS and CNX (control for a type I membrane protein) was digested in the absence or presence of SDS and no longer recognized by an anti-C terminal antibody.