Supplementary Figure 2: Compromised cell surface expression of PKD1^S99I.

Expression levels of several pathogenic mutants of PKD1 in transiently transfected HEK293T cells. Cells were transfected with human PKD1 cDNA tagged at its C-terminus with 3xHA (hPKD1-HA, lane 1), hPKD1^C47S-HA (lane 2), hPKD1^S75F-HA (lane 3), hPKD1^S99I-HA (lane 4), hPKD1^W139C-HA (lane 5), or pcDNA3 (mock, lane 6). HA-tagged wild type or mutant PKD1 was immunoprecipitated with a mouse α-HA and complexes were immunoblotted with a rabbit α-HA. (b) Compromised cell surface expression of PKD1^S99I. HEK293T cells were transfected with (GFP, mock, lane 1), wild type PKD1 (PKD1^WT) plus TRPP2 (lane 2) or PKD1^S99I plus TRPP2 (lane 3). Cells were labeled with cell impermeant biotin, lysed, and biotinylated proteins were captured with streptavidin beads. Biotinylated proteins were immunoblotted by α-PKD1 (7e12, upper panel). Expression levels of PKD1 constructs in lysates are shown in lower panel. (c) Cell surface expression of TRPP2 is not affected by PKD1^S99I. Biotinylated proteins (prepared as described in b) were immunoblotted by α-TRPP2 (G20, 1:500, Santa Cruz Biotechnology) (upper panel). Expression levels of TRPP2 in total lysates are shown in lower panel. TRPP2 migrated as a high molecular weight species (aggregated form) and a monomeric species (∼100 kDa), because lysates were boiled for 5 min, before loading onto SDS PAGE. (d) Pkd1^+/+ or Pkd1^−/− MEFs were labeled with cell impermeant biotin and biotinylated proteins were immunoblotted with α-TRPP2 (G20, 1:500, lanes 2 and 3). Expression levels of TRPP2 in Pkd2^−/−, Pkd1^+/+, and Pkd1^−/− cells are shown in lanes 1, 4, and 5, respectively. Note that a non-specific band detected by α-TRPP2 (indicated by an asterisk) is only present in total cell lysates, but not in the biotinylated protein pool. Experiments were successfully repeated 3 times. Uncropped western blots are provided in Supplementary Fig. 8.