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. 2006 Aug;26(16):5994–6004. doi: 10.1128/MCB.01630-05

FIG. 3.

FIG. 3.

Ro52α interacts with Skp2 and βTrCP. (A) HA-Ro52α and MT-Skp2 were expressed individually or together in HeLa cells, and aliquots of lysates were either subjected to immunoprecipitation with anti-HA 12CA5 antibody followed by immunoblotting with rabbit anti-Skp2(fl) antibody (upper panel) or directly processed for immunoblotting with anti-HA MAb HA11 (middle panel) or anti-Myc MAb 9E10 (lower panel). (B) The panel shown is as described for panel A, except that HA-Ro52α(ΔR) lacking the RING domain was transfected instead of HA-Ro52α(wt). (C) In vitro translates (IVTs) of FT-βTrCP1, FT-βTrCP2, or pVHL were incubated with MBP (lanes 5, 7, and 9) or MBP-Ro52 (lanes 4, 6, and 8) fusion proteins, and bound proteins were analyzed by SDS-PAGE (lanes 2 through 4). Lanes 1 to 3, input IVTs. −, presence of; +, absence of. (D) FT-mPIAS3L, FT-βTrCP1, or FT-βTrCP2 was expressed in HeLa cells, together with HA-Ro52α, and aliquots were subjected to immunoprecipitation with anti-Ro52 MAb and processed for immunoblotting with anti-FT antibody M2 (upper panel) or anti-Ro52 MAb (lower panel). Other aliquots were directly processed for immunoblotting (whole-cell extracts [WCE]) using anti-Ro52 and anti-FT M2 MAbs. The left arrowhead indicates the migration of FT-mPIAS3L (negative control), while the right arrowheads indicate the positions of FT-βTrCP1 and FT-βTrCP2. (E) IVTs of MT-Skp2, MT-Skp2(ΔLRR), or MT-Skp2(ΔF30) were incubated with MBP (lanes 5, 7, and 9) or MBP-Ro52 (lanes 4, 6, and 8) fusion proteins, and bound proteins were analyzed by SDS-PAGE (lanes 2 to 4). Lanes 1 through 3, input IVTs. (F) HeLa cells were transfected with siRNAs corresponding to either a nonrelevant mRNA (control [ctrl]) (lanes 1 and 2) or Skp1 mRNA (lane 3). Aliquots were processed for immunoprecipitation with either control IgG (lane 1) or anti-Ro52 MAb (lanes 2 and 3) that had been covalently coupled to protein A-Sepharose beads using dimethylpimelimidate and processed for immunoblotting using anti-Skp2 MAb mix or anti-Ro52 MAb 83.34. Other aliquots were directly processed for immunoblotting with antibodies directed against indicated proteins.