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Fig. 2. Components of the sumoylation system are expressed in basal and differentiated HaCaT cells. RNA (A-C) or protein (D,E) were harvested from HaCaT cells maintained in either low [Ca²⁺] (0.03 mM) or high [Ca²⁺] (2.8 mM) medium. Harvesting and analysis were performed as described in Materials and Methods. (A) Quantitative RT-PCR of involucrin mRNA levels. (B) RT-PCR analysis of the indicated sumoylation system genes. (C) Quantitative RT-PCR analysis of the relative levels of Ubc9, SAE1, SAE2, SUMO1, SUMO2 and SUMO3 mRNAs. (D) Immunoblots for SAE1, Ubc9 and -tubulin. SAE1 and Ubc9 blots were quantified by densitometry and normalized to -tubulin. The graph shows the relative protein levels for HaCaT cells maintained in low and high [Ca²⁺] medium, and the lower panels show representative SAE1 and Ubc9 immunoblots with their corresponding -tubulin controls. (E) Anti-SUMO immunoblot of total cell extracts from HaCaT cells maintained in either low or high [Ca²⁺] medium. Protein concentrations in the extracts were normalized to -tubulin, and the samples were resolved by 10% SDS-PAGE. Arrows indicate sumoylated proteins whose levels appear higher in the high [Ca²⁺] culture. Quantitative results in A, C and D are the average of at least three independent experiments.

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