Figure 3. Evaluation of the feasibility of QDB analysis at cellular level.

(A) Characterization of anti-p65 antibody. Insert, the specificity of anti-p65 antibody was investigated using cellular lysate prepared with HEK293 cells in Western blot analysis. The whole membrane was scanned using a blot scanner from Li-Cor to ensure only one band with expected molecular weight was observed. A. The whole cell lysate was serially diluted as indicated in the figure, and applied to the QDB plate at 2μL/unit. The plate was processed as described in the Materials and Methods, and the results were analyzed with simple linear regression analysis with R² at 0.992. (B) Comparison of the relative p65 levels between Luciferase and p65 clones. HEK293 cells were transfected with ShRNA-p65 or ShRNA-Luciferase respectively using Fugene 6 transfection reagent. Stable clones were selected using puromycin at 5 μg/mL from cells transfected with ShRNA-p65 (p65 clones) or ShRNA-Luciferase (Luciferase clones) until they were visible under naked eyes. Clones were picked up and transferred to two 96 well plate at 1:9 ratio (plate A and B for description purpose). Luciferase clones were labeled as L1 to L5, while p65 clones were labeled sequentially. The 96 well plates with a larger portion of cells (plate A) were allow to grow for one or two days before they were collected to prepare for cell lysate as described in the Materials and Methods. The whole cell lysate from individual clone was used for QDB analyses of tubulin and p65 levels, and the relative level of p65 (ratio of p65 level over tubulin level) was used to compare endogenous p65 expression levels in each clone, using the average of the p65 expression levels in luciferase clones as 1. The results were averaged to compare endogenous p65 levels between luciferase and p65 clones at population level. *, p< 0.05 using student T-test. (C) The relative p65 level of all 76 clones, including 5 luciferase clones and 71 p65 clones, was plotted individually. The result presented is the average of three independent experiments in triplicates. (D) The QQ plot analysis of the distribution of the endogenous p65 levels of 71 p65 clones is shown to demonstrate the normal distribution of relative p65 level among p65 clones. (E) Representative clones were picked up from the plates with less cells (plate B) based on the results shown in Figure 3C, and transferred to 60 mm dishes to allow them to reach sufficient cell numbers for Western blot analysis using anti-p65 and anti-tubulin antibodies (Lower panel). The results of QDB analyses of these representative clones from Figure 3C were re-plotted in the upper panel for comparison purpose.