Figure 1.
Protein level affects epitope detection.
(A) A fixed amount of purified cyclin B1 (6.36 ng) was added to a serial dilution of trypsin inhibitor (TI) and subjected to electrophoresis and Western blotting. (B) Relative intensity of the cyclin B1 bands is presented corresponding to trypsin inhibitor levels. Cyclin B1 intensity was maximal at 25–50 µg trypsin inhibitor and decreased at lower and higher protein levels. Densitometry readings from 3 experiments and 6 gels were combined by normalizing by linear regression on measurements common to each paired series. The number of common points ranged from 6 to 8. The combined set was then normalized to the 500 TI mean value, therefore the Y axis can be read as fold over this value.
Figure 2.
Extraction of cyclin B1 in various lysis buffers.
Equal numbers of dissociated PC3 cells were solubilized in lysis buffer containing RIPA, Triton X-100 or SDS (see Materials and Methods) and analyzed for cyclin B1 by Western blot (A). Increasing the SDS level to 23% did not release more cyclin B1 (B). There was some variability between lanes but, based on multiple gels, it was clear that cyclin B1 was readily extracted by all methods.
Figure 3.
Manipulation of cyclin B1 levels.
Lysates were made from known numbers of PC3 and DU145 cells that had either been grown to confluence or harvested at very low density. Equal numbers of cells were loaded in each lane and Western analysis of cyclin B1 was performed. The increase in total cyclin B1 in subconfluent relative to confluent cells was 4.4 fold for PC3 cells and 2.5 fold for DU145 cells (A). Nocodazole (NDZ) treatment for the indicated hours produced a final 17.6 fold increase over confluent cells for DU145 cells (B) and a 52 fold increase for PC3 cells (C).
Figure 4.
Comparison of MeOH fixed cells to MeOH fixed-lyophilized cells.
A culture of PC3 cells was trypsinized, washed, and then divided into three equal parts. One part was solubilized for electrophoresis, one part was fixed with methanol, and the third part was fixed with methanol and then lyophilized. The methanol-fixed and lyophilized cell preparations were washed then solubilized with 23% SDS. The lanes were loaded with equal numbers of cells.
Figure 5.
Cytometry and quantitative Western blots of standards.
(A) Left 4 lanes are recombinant cyclin B1 at two-fold dilutions. Recombinant cyclin B1 and lysate concentrations were adjusted so that the band intensities for recombinant cyclin B1 spanned the range of those of the lysates. Trypsin inhibitor was added to each sample to bring the total protein per lane to 50 µg. (B) Lyophilized standard cell preparations from the same samples as used for Western blot were stained for cyclin B1 with GNS1-Alexa Fluor 647 (A647) and DNA (DAPI). Arrows indicate the mean cyclin B1 fluorescence. Background fluorescence was subtracted and data were plotted as a hyperlog transformation which allows plotting of negative and zero values on a linear lower section (−10 to 10) and higher values (>10) logarithmically [22].
Table 1.
Standard Curve.
Figure 6.
Correspondence between cyclin B1 measured by cytometry and by Western blotting.
Average cyclin B1 levels were calculated from luminescence measurements of imaged Western blots then plotted versus mean fluorescence values (representative data shown in Figure 5). (A) Means and the 95% confidence interval are plotted. Cytometry was performed in duplicate. Western blots varied from 3 to 6 replicates. Individual values are plotted in (B).
Figure 7.
Samples of HeLa, K562, and RKO were fixed with methanol then stained for cyclin B1 with GNS1-AlexaFluor 647 (A647) and DNA (DAPI). Part of the sample was also lysed and subject to Western blot analysis in the same manner as the standard lysates described in Figure 5. (A) cytometry data are displayed as in Figure 5. Arrows indicate the mean cyclin B1 related fluorescence. (B) Western blot prepared as in Figure 5. The lanes for the test cell lines were not loaded with equal cell numbers; loading was adjusted to obtain bands with intensities within the range of the cyclin B1 standards. Protein concentration was equalized with trypsin inhibitor. (C) co-plot of the standard curve and the test samples using the measured values for cyclin B1 molecules obtained by Western analysis for the test samples. HeLa = red circles; K562 = blue circles; RKO = green circles. The 95% prediction band for the standard curve is plotted (dashed lines). (D) Molecules were determined by Western blotting (Y axis) and by cytometry using the standard curve (X axis). The dashed line is a one-to-one correlation. Color coding is as in C.
Figure 8.
Cell cycle expression of cyclin B1.
The cytometric data for HeLa, K562, and RKO cells (Figure 7) were gated to exclude endoreduplicated or binucleate cycling cells (4C 8C); mitotic cells after metaphase, and outliers. The purpose was to expose only the cells distributed about the two dimensional center as a function of DNA content. These data represent the most common cycling cell in the population. Arbitrary regions were set, moving through the data from early G1 through late G2+M (A–C). The mean cyclin B1 levels from each region are plotted as a function of the cell frequency in the region (D–F). The frequency data were renormalized from the first non-zero cyclin B1 point and plotted to compare the committed part of the cell cycle (cyclin B1 is detectable) for each cell line (G). In A-G, the X axis scale is 0–1, and the Y axis scale is 0–4×106.
Table 2.
Comparison of Cyclin B1 Quantification.