Figure 3.

Reduced Steady-State Levels of TFIIE and Normal Levels of Other General Transcription Factors in Primary Fibroblasts from Affected Individuals TTD379BE and TTD28PV

(A) Immunoblot analysis of whole-cell lysates with antibodies against the α and β subunits of TFIIE, TBP, TFIIB, the CDK7, p44, p62, and XPD subunits of TFIIH, and TTDN1. γ-tubulin was used as loading control. The amount of each protein was first expressed as the mean value of the levels observed in the three increasing concentrations of the cell lysate and normalized to the γ-tubulin content. The protein levels in both TTD cell strains were then expressed as percentages of the corresponding values in the normal (C3PV) cells. The reported values are the means of at least two independent experiments. Bars indicate the SE.

(B) Anti-TFIIEα and anti-TFIIEβ immunoblot analysis of whole-cell lysates from lymphoblastoid cells of TTD28PV and her unaffected mother, father, and sister. The amount of the analyzed proteins was determined as described in (A); the levels of TFIIEα and TFIIEβ are expressed as percentages of the corresponding values in the normal C6PV lymphoblasts. The mean levels of two independent experiments are reported. Bars indicate the SE.

(C) Reduced immunofluorescence of TFIIEβ protein in TTD379BE cells (incubated with 2 μm beads, red arrow) compared to normal cells (incubated with 1 μm beads, yellow arrows).

(D) Immunofluorescence detection of reduced levels of TFIIEβ and TFIIEα in TTD28PV and TTD379BE cells. TTD and normal cells were labeled as in Figure 2C and irradiated with 100 J/m² UVC through a filter with 5 μm pores to generate localized DNA damage. The TFIIE proteins were reduced in the TTD cells. There was no localization of TFIIE proteins at the site of localized DNA damage in the TTD or normal cells.