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. 2013 Jan;33(1):14–27. doi: 10.1128/MCB.00887-12

Fig 1.

Fig 1

AKAP-Lbc anchors IKKβ. (A) Pulldown experiments were performed by incubating GST or the GST-AKAP fragment encompassing residues 1923 to 2336 with mouse heart extracts. Proteins were resolved by SDS-PAGE and silver staining and identified by MS/MS spectrometry. (B) Pulldown experiments were performed as indicated in panel A. IKKβ binding was detected by immunoblotting (IB) (top). GST fusion proteins were visualized by Ponceau S staining (bottom). (C) GST and the GST-AKAP-Lbc-1923-2336 fragment were used as bait in pulldown experiments with increasing concentrations of the purified S-tagged 307-756 fragment of IKKβ. Binding of the S-tagged IKKβ fragment was detected by immunoblotting (top). GST fusion proteins were visualized using Ponceau S staining (bottom). (D) Lysates from HEK293 cells transfected with HA-IKKβ in combination with control vector or the plasmid encoding Flag-AKAP-Lbc were immunoprecipitated (IP) using anti-Flag antibodies. Proteins in the lysates and immunoprecipitates were identified by immunoblotting using antibodies against the HA tag or the Flag tag as indicated. (E) Lysates from HEK293 cells transfected with AKAP-Lbc-GFP in combination with control vector or the plasmid encoding Flag-IKKβ were immunoprecipitated using anti-Flag antibodies. Proteins in the lysates and immunoprecipitates were identified by immunoblotting using antibodies against GFP or the Flag tag as indicated. (F) Rat NVMs were serum starved for 24 h and treated with 10−4 M PE for the indicated periods of time. Extracts were subsequently subjected to immunoprecipitation with either nonimmune IgGs or affinity-purified anti-AKAP-Lbc antibodies. Western blots of the immunoprecipitates and the cell extracts were revealed using either anti-IKKβ (top) or affinity-purified anti-AKAP-Lbc polyclonal antibodies (bottom). (G) Rat NVM extracts were subjected to immunoprecipitation as described for panel F. Western blots of the immunoprecipitates and the cell extracts were revealed using either anti-RhoA monoclonal antibodies (top) or affinity-purified anti-AKAP-Lbc polyclonal antibodies (bottom).