Journal: Cells

Article Title: Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process

doi: 10.3390/cells11111832

Figure Lengend Snippet: ATAD5 URM is important for efficient PCNA unloading. ( A ) RFC2-5 binding was not affected by URM mutations. URM mutants were transiently expressed in 293T cells. To monitor the RFC2-5 binding, FLAG-immunoprecipitation was performed and co-isolated RFC4 was analyzed by immunoblot. The amount of co-purified RFC4 was not changed by URM mutations. ( B ) Coomassie blue stained SDS-PAGE of URM mutant ATAD5 (812–1844)-RLC. URM mutants formed a pentameric complex with RFC2-5. ( C ) URM mutants are defective in PCNA unloading in vitro. Top —in vitro PCNA unloading assay was performed with URM mutants. Compared to wild type, URM mutants showed defects in releasing PCNA from DNA. Bottom —quantification of the relative PCNA amount on DNA. Error bars indicate standard deviation ( n = 2).

Article Snippet: The following antibodies were used: anti-PCNA (Rabbit), anti-LaminB1 antibodies (Abcam, Cambridge, UK); anti-FLAG, anti-HA antibodies (Merck, Darmstadt, Germany); anti-PCNA (mouse), anti-RFC4 antibodies (Santa Cruz, Dallas, TX, USA); anti-Myc antibody (Merck, Darmstadt, Germany).

Techniques: Binding Assay, Immunoprecipitation, Isolation, Western Blot, Purification, Staining, SDS Page, Mutagenesis, In Vitro, Standard Deviation

Journal: Cells

Article Title: Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process

doi: 10.3390/cells11111832

Figure Lengend Snippet: ATAD5 C-terminus is required for efficient PCNA unloading. ( A ) ATAD5 C-terminus is conserved among species. Primary sequences of ATAD5 C-terminus from different species are aligned. ( B ) ATAD5 C-terminus is required for stable RFC2-5 binding. ATAD5 (812–1844) or ATAD5 (812–1799) were transiently expressed and FLAG-immunoprecipitation was performed. The amount of co-precipitated RFC4 was reduced with ATAD5 (812–1799). ( C ) ATAD5- C-terminus is essential for PCNA unloading. ATAD5 (812–1844) or ATAD5 (812–1799) were transiently expressed in ATAD5 depleted cells. After chromatin fractionation, the amount of chromatin-bound PCNA was monitored by immunoblot. Chromatin-unbound soluble fractions were shown to examine total amount of each protein. ATAD5 (812–1799) failed to reduce PCNA level on chromatin compared to ATAD5 (812–1844). Number below the PCNA blot indicates relative amount of chromatin-bound PCNA.

Article Snippet: The following antibodies were used: anti-PCNA (Rabbit), anti-LaminB1 antibodies (Abcam, Cambridge, UK); anti-FLAG, anti-HA antibodies (Merck, Darmstadt, Germany); anti-PCNA (mouse), anti-RFC4 antibodies (Santa Cruz, Dallas, TX, USA); anti-Myc antibody (Merck, Darmstadt, Germany).

Techniques: Binding Assay, Immunoprecipitation, Fractionation, Western Blot