Fig 1.
Establishment of stable cell lines expressing CSB-TAP protein and Identification of proteins that co-purified with CSB-TAP.
(A) Western blot showing the expression of endogenous CSB full-length (CSB fl) and CSB-PGBD3 proteins in wild type MRC5 fibroblasts and either chimeric CSB-TAP protein (CSIAN TAP/CSB) or TAP domain (CSIAN TAP) in CSB-deficient fibroblasts CSIAN after stable transfection. TAP tagged proteins were detected using rabbit polyclonal anti TAP tag (CAB 1001, Pierce) and endogenous CSB, either fl and PGBD3 isoforms, using rabbit polyclonal anti-ERCC6 (H300, Santacruz). (B) UV survival demonstrated the complementation of UV survival in CSB-TAP transfected cells. Clonogenic survival after UV exposure in MRC5, CSIAN and isogenic clonal populations of CSIAN transfectant cell lines is shown as the percentage of survival. Results shown are the average values of three independent experiments. (C) Silver staining of proteins associated with CSB-TAP and TAP that were isolated by tandem affinity purification and, separated on a 4–12% Bis-Tris gel.
Fig 2.
Names of proteins co-purifying with CSB-TAP fusion protein and their associated biological processes.
Fig 3.
Interactome (protein-protein interaction) of CSB-associated proteins in CSIAN cells.
The protein-protein interaction network was constructed using String software online (version 8.3, www.string-db.org) for CSB-TAP co-purifying proteins. Blue dotted line area represents the RNA splicing cluster. The red dotted line area represents the NHEJ repair cluster; the green dotted line area represents the gene expression regulators cluster and the grey dotted line area the proteasome cluster.
Fig 4.
Co-immunoprecipitations studies.
Co-immunoprecipitation was performed using the lysates of CSIAN cells, transiently expressing flag tagged CSB and myc-tagged proteins of interest. Whole cell extracts, prepared 24 hr post transfection, were immunoprecipitated for either CSB or putative interacting proteins using respectively Flag or c-Myc monoclonal antibody conjugated agarose resins, followed by Western blot analysis using anti-myc and anti-flag antibodies respectively. * two isoforms p72 and p82 arise from ddx17 gene through the use of different in-frame translation initiation codons. (WCE, whole cellular extract; FT, Flow-Through; IP, immunoprecipitation).
Fig 5.
Co-immunoprecipitations studies.
Co-immunoprecipitation was performed using the lysates of CSIAN cells, transiently expressing flag tagged CSB and myc-tagged proteins of interest. Whole cell extracts, prepared 24 hr post transfection, were immunoprecipitated for either CSB or putative interacting proteins using respectively Flag or c-Myc monoclonal antibody conjugated agarose resins, followed by Western blot analysis using anti-myc and anti-flag antibodies respectively. (WCE, whole cellular extract; FT, Flow-Through; IP, immunoprecipitation).
Fig 6.
Co-immunoprecipitations studies.
Co-immunoprecipitation was performed using the lysates of CSIAN cells, transiently expressing flag tagged CSB and myc-tagged proteins of interest. Whole cell extracts, prepared 24 hr post transfection, were immunoprecipitated for either CSB or putative interacting proteins using respectively Flag or c-Myc monoclonal antibody conjugated agarose resins, followed by Western blot analysis using anti-myc and anti-flag antibodies respectively. (WCE, whole cellular extract; FT, Flow-Through; IP, immunoprecipitation).
Fig 7.
Schematic diagram illustrating the impact of functional loss of CSB on a multitude of biological processes with special relevance to some of the pathological symptoms observed in the CSB patients.
Some of the pathological symptoms presumably arising due to deficiencies in various biological processes (RNA metabolism, Chromatin remodeling, DSB repair, proteasome and signalosome mediated cellular activities) due to CSB loss are indicated (blue). The double-headed arrow indicates the known interactions between signalosomes and proteasomes.