Knockdown of UbcH5, UbcH6, or UbcH7 mRNA reduces TdT ubiquitylation.

<p>(A and B) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH5a or UbcH5c using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h. mRNA levels of UbcH5 isoforms were measured by real-time PCR, normalized to GAPDH, and are expressed as their ratio to cells transfected with a control siRNA (A). Ubiquitylated Myc-TdT and UbcH5 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH5, or anti-actin antibody (B). (C) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH6 or UbcH7 using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. Ubiquitylated Myc-TdT, UbcH6, and UbcH7 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH6, anti-UbcH7, or anti-actin antibody.