CaMKIV accumulates during differentiation of monocyte-derived dendritic cells. (A) CD14⁺ mononuclear cells were cultured in the presence of GM-CSF and IL-4. Whole-cell lysates were prepared at the indicated times and analyzed by immunoblot with specific antibodies (CaMKIV and actin). Aliquots of cells were used to measure CaMKIV and actin mRNA levels by quantitative reverse transcription–polymerase chain reaction. Bottom panel shows mean (± SD) of optical density measurements expressed as the ratio between the CaMKs and actin bands (n = 4). *P < .01. (B) Calpain regulates CaMKIV accumulation in differentiating monocytes. CD14⁺ mononuclear cells were cultured in the presence of ALLM, a selective calpain inhibitor (ALLM), or GM-CSF and IL-4 and analyzed for CaMKIV expression by immunoblot (top). The bottom panel shows mean (± SD) of the optical density measurements expressed as the ratio between the CaMKIV and actin bands (n = 4). *P < .01. (C-E) Intracellular distribution of CaMKIV in differentiating monocytes. (C) Transmission and confocal fluorescent immunocytochemistry images of CaMKIV expression in monocytes cultured for: 2 hours in regular medium (i,ii); 2 hours in the presence of GM-CSF/IL-4 or ALLM (iii, iv, v, and vi, respectively); and 120 hours in the presence of GM-CSF/IL-4 (vii,viii). (D) Line profiles of cells indicated by white arrows in the corresponding subpanels in panel C. The line segment is 20 μM; F indicates the fluorescence intensity in arbitrary units (a.u.). The bottom right graph shows the ratio (R) between the mean fluorescence intensity of Alexa Fluor 594 (CaMKIV) and Hoechst 33342 (nuclear staining) in the nuclear region along different line profiles. Means (± SD) represent 20 independent line profiles. *P < .01. (E) Expression and line profile analysis of CaMKIV in monocytes treated for 120 hours with GM-CSF/IL-4..