Figure 1. Knockout of ORC2 in HCT116 p53-/- cells.
(A) Strategy for insertion of a blasticidin gene and poly A site in the fourth exon of ORC2 at aa 40 of ORC2. (B) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC2+/+. ORC2 Knockout clones, B2 and BP8 have an insert on both alleles of ORC2 as indicated by the absence of 0.6 kb PCR product. (C) Verification of antibodies recognizing N-terminal or C-terminal parts of ORC2. Recombinant ORC2 protein halves with Flag epitope tags were expressed and blotted with indicated antibodies. Ponceau S staining of total protein shows equal loading of lanes.* indicates full length endogenous ORC2 protein. Arrow indicates recombinant protein. (D) Quantitative Western blot for ORC2 with an antibody recognizing the N-terminal half of ORC2. Indicated amount of lysate loaded in each lane. (E) Western blot with antibody recognizing C-terminal half of ORC2. * Non specific band (F) Input cell lysate and immunoprecipitates of ORC2 immunoblotted for ORC2. Darker exposure of the top blots is shown in the middle. HSP90 in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. (G) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC2 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. (H) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. Ponceau S staining of histones serves as loading control for chromatin fractions. For each panel, all the lanes are from the same blot and exposure. (I) Comparison of Coomassie Brilliant Blue signal of pure BSA and recombinant purified GST-ORC2 to show that the top-most band in the ORC2 lane is at 170 ng/ 10 μl. (J) Immunoblot with different amounts of cell lysate with the GST-ORC2 to show that 1×10e5 cells give an ORC2 signal equal to 2.54 ng (1.4 fold of 1.67 ng) of GST-ORC2, which corresponds to 153×10e8 molecules of GST-ORC2. (K) Western blot of ORC2 in HBEC and 293T cell lines. Ponceau S staining of total protein or immunoblot of Chk1 show equal loading of the pairs of lanes.