Overexpression of HLTF in K562 cells and its effect on the β-globin transcription.

(A) Western blot of total protein extract of K562 cells stably transfected with pCIneo vector alone and pCIneo containing HLTF with poly His tag at its N-terminal end. Protein (70 μg) was loaded in each lane. The cell-free extracts are indicated at the top of the panel, and standard molecular weight marker positions are indicated at the left-hand side. The arrows mark points at the HLTF band. The antibodies against HLTF and poly His used to probe the blot are mentioned at the bottom of the panel. In the blot probed with the anti-His antibody, multiple bands found below the 115-kd HLTF band are common to both the lanes. These bands presumably are due to the binding of anti poly-His antibody to proteins containing short stretches of histidines. (B) Transient transfections of −136 to +54 bp β-promoter (cloned into theHindIII-XhoI site of the pGL3) into normal and HLTF-overexpressing cells. (C) Transient transfection of pGL3 constructs containing β-promoter and its mutant variants and HS2 at the enhancer site. These constructs are described in Figure 5. The values in the bar diagram are the averages of 12 experiments, including 3 separate experiments performed in duplicate with each of the 2 separate pools of K562 cells stably transfected with HLTF. The luciferase activities are normalized as activity per 10 μg total protein. Horizontal lines above each bar indicate 1 SD in the average of the experimental values.