Diffusion coefficient of eGFP-Cdc45 at G1 to S phase transition and in S phase and after UV damage.

<p>Mean and standard deviations obtained from at least 15 cells.</p

HeLa S3 cells stably expressing eGFP-Cdc45.

<p>Panel a, schematic diagram of eGFP-Cdc45 protein encoded by Cdc45L ORF cloned into pIC113gw vector. Panel b, total cell extract HeLaS3 cells (-ve) and Hela S3 cells stably expressing eGFP-Cdc45 (eGFP-Cdc45) normalized for protein content and analysed by western blotting using antibodies raised against Cdc45 and β-Actin, which serves as a loading control. Panel c, western blot analysis of immunoprecipitation of eGFP-Cdc45 using GFP-Trap IP from HeLa S3 cells transiently expressing eGFP-Cdc45. Verification of purification of eGFP-Cdc45 and co-immunoprecipitation of Mcm7 was carried out using antibodies raised against Mcm7 and Cdc45. Input (L), unbound (FT), mock IP (-ve) and IP from cells expressing eGFP-Cdc45 (eGFP) indicate yield of the IP and co-immunoprecipiation. Antibody light chain acts as a loading control.</p

Association of Cdc45 with chromatin synchronized HeLa S3 cells and after DNA damage.

<p>Panel a, chromatin-associated lysate from 1×10⁶ Hela S3 cells synchronized at various cell cycle stages by two consecutive thymidine block analysed by western blotting using antibodies raised against Cdc45, Mcm7, Mcm5, Lamin B1, P261 and P125 of Pol ε and <i>δ</i>, respectively. The latter serves as a loading control. Asynchronous control cells (Asn) or cells analysed at times ranging from 0 to 12 h following release from the second thymidine block (TdR 0 to TdR 12) were analysed in parallel by FACS. Corresponding FACS profiles for relevant timepoints are also shown. Panel b, western blot of chromatin-associated Cdc45 following UVC treatment. HeLa S3 cells treated with 5 J/m² UVC harvested at indicated timepoints post treatment with untreated cells (UT) acting as a control. Chromatin-associated lysates normalized for protein content were analysed by western blotting using antibodies raised against Cdc45 and Lamin B1, which serves as a loading control.</p

Electrophilic Reactivity of Tetrabromorhodamine 123 is Bromine Induced: Convergent Interpretation through Complementary Molecular Descriptors

Nucleophilic addition of water and of methanol to 3,6-diamino-2,4,5,7-tetrabromo-9-[2-(methoxycarbonyl) phenyl]-9<i>H</i>-xanthen-9-ylium, 4BrR123, yields respectively 2-(3,6-diamino-2,4,5,7-tetrabromo-9-hydroxy-9<i>H</i>-xanthen-9-yl)­xanthyl benzoate, HO4BrR123 and 2-(3,6-diamino-2,4,5,7-tetrabromo-9-methoxy-9<i>H</i>-xanthen-9-yl)­xanthyl benzoate, MeO4BrR123. The novel experimental results are addressed theoretically. The linear free energy relationship, LFER, second-order perturbation theory analysis of the natural bond orbital, NBO, and quantum theory of atoms in molecules, QTAIM, lead to the same conclusion: the electron-withdrawing effect of bonded Br atoms in 4BrR123 extremely enhances the molecular electrophilicity, as compared to 3,6-diamino-9-[2-(methoxycarbonyl) phenyl]-9<i>H</i>-xanthen-9-ylium, R123. The reactivity of these diaminoxanthylium cations is discussed in the context of local and global softness in extended conjugated systems

Auto-correlation curves of eGFP-Cdc45.

<p>The figure shows typical auto-correlation curves of eGFP-Cdc45 (□) in asynchronous HeLa S3 cells stably expressing eGFP-Cdc45. In the upper panel the solid black line corresponds to a two-component free diffusion model and in the lower panel the gray line is the residual of the fit.</p

Analysis of the size distribution of Cdc45-containing protein complexes during the cell cycle and after UV damage.

<p>Asynchronous (Asn) UVC-treated (+UVC, 5 J/m², 1 h post-treatment), G1/S transition or S phase synchronized HeLa S3 cells stably expressing eGFP-Cdc45 were lysed and normalized for protein content and separated by gel filtration chromatography analysed by western blotting using antibodies raised against Cdc45, Mcm5 and RPA 32. (panels a, b, c, and d, respectively). Theoretical molecular weight (kDa) and Stoke's radius (Å) of protein standards are overlayed. RPA32 acts as a marker for DNA damage response following UVC treatment. FACS analysis is provided for asynchronous (Asn), G1/S transition and S phase synchronized cells (e) and asynchronous cells treated with 5 J/m², 1 h post-treatment (f).</p