BMCC1 Is an AP-2 Associated Endosomal Protein in Prostate Cancer Cells
Figure 2
BMCC1 protein interaction mapping.
A. Purification of BMCC1 interacting proteins. Crosslinked BMCC1 GST fragment 3 was used as bait to purify interacting proteins. GST crosslinked resin is a non-specific binding control. Interacting proteins were analysed by silver-stained SDS-PAGE. BMCC1-F3 interacting proteins were excised from the silver stained gel, processed and identified by MALDI TOF/TOF. Band identities are indicated.
B. Immunopurification of BMCC1 and interacting proteins. Sheep anti-BMCC1 Ab-4 or nonspecific (NS) sheep IgG were incubated with LNCaP total lysate overnight and complexes were precipitated with protein G agarose. Immunoprecipitates were subjected to immunoblot using antibodies specific for BMCC1 (Ab-1), β-adaptin and AP2A1/2 as indicated. IgG (loading) indicates equal antibody input.
C. Immunopurification of BMCC1 and interacting proteins, using MALDI MS/MS identification. 80% confluent T-75s of LNCaP cells were harvested 3 days post-transfection with control siRNA (siControl) or BMCC1 siRNA (siBMCC1). Clarified lysates were immunoprecipitated with Ab-4 and analysed by Coomassie stained 5% gel or western blotting duplicate 5-15% gels for AP-2A1/2 and β-adaptin. IgG (loading) indicates equal antibody input. Immunoprecipitated BMCC1 was excised from the Coomassie gel and its identity was confirmed by MALDI MS/MS (this particular Coomassie gel has been Gamma-curve adjusted for visual clarity and corresponds to MALDI data Sample A in Supp Table 4).