ID4 methylation and protein expression in CLL cells. Top, Immunoblot analysis of ID4 expression in normal B cells or CLL cells. Lysate from HEK 293 T cells transfected with vector alone or ID4 cDNA were included as controls. Tubulin expression served as a loading control. Blots shown are representative of a total of 8 samples for each normal and CLL CD19⁺ B cells, all run in duplicate. Raw intensities of ID4 and tubulin were background-corrected and ratios of ID4 over tubulin were log2-transformed for each sample. Analysis of variance model was used and each immunoblot was treated as a block. Differences of ID4 levels between CLL and normal were evaluated by 2-sided t test. ID4 expression in CLL cells is 0.35-fold the level in normal B cells (P = .0002). Bottom, Correlation between protein expression and methylation level of ID4 was calculated by Pearson correlation coefficients. On the basis of the results obtained from 8 healthy and 8 CLL B-cell samples, the correlation coefficient is −0.53 (P = .034) between average methylation across all amplicons and ID4 protein expression.