Downregulation of NELF protein during granulocytic differentiation. (A) Western blot of NELF subunits from different days during granulocytic differentiation of human CD34⁺ HSPCs. GAPDH serves as a loading control. Quantification results are in supplemental Figure 1C. (B) qRT-PCR of messenger RNA (mRNA) levels of NELF genes during granulocytic differentiation of CD34⁺ cells. Gene expression is normalized to β-actin and presented as fold change (FC) relative to day 0 (n = 3; mean ± standard error of the mean [SEM]). (C) Western blot of NELF subunits on differentiation day 3. MG132 treatment was done by adding 0.2 μM MG132 overnight before harvesting cells for protein extraction. Quantification results are in supplemental Figure 1D. (D) Western blot of NELF subunits during neutrophil differentiation of mouse 32Dcl3 cell line. Quantification results are in supplemental Figure 1E. (E) qRT-PCR of mRNA levels of Nelf genes during neutrophil differentiation of mouse 32Dcl3 cells. Gene expression is normalized to β-actin and presented as fold change relative to day 0 (n = 3; mean ± SEM). (F) Western blot of NELF-A and -E subunits from cells isolated from mouse bone marrow (BM). NA, NELF-A; NB, NELF-B; NC/D, NELF-C/D; NE, NELF-E.