UBAP2L does not affect BMI1 and RNF2 protein levels, and p16^Ink4a expression, and forms a distinct PcG complex with BMI1. (A) Effect of Ubap2l silencing on BMI1 and RNF2 protein levels in Lin⁻ cells. Following infection of Lin⁻ cells with shUbap2l, GFP⁺ cells were sorted. Ubap2l knockdown was determined by qRT-PCR (left panel). The values for Ubap2l mRNA levels are expressed relative to Hprt with the standard error of the mean shown and the value for shLuc was set to 1. BMI1 and RNF2 protein levels were monitored by western blotting (right panel). (B) Effect of Ubap2l deletion on BMI1 and RNF2 protein levels determined by western blotting in Ubap2l^+/+, Ubap2l^+/−, and Ubap2l^−/− MEFs. (C) Impact of Ubap2l knockdown on p16^Ink4a levels. Lin⁻SCA1⁺ cells were infected with shUbap2l or shBmi1 and the mRNA levels of p16^Ink4a were determined by qRT-PCR immediately after infection. For all genes, the values are expressed relative to Hprt and the value for shLuc was set to 1. The average of 2 infections is shown with the standard error of the mean. Representative of 2 independent experiments. (D) Effect of Ubap2l deletion on p16^Ink4a levels. p16^Ink4a mRNA levels were determined by qRT-PCR in Ubap2l^+/+ and Ubap2l^−/− MEFs. The values are expressed relative to Hprt and the value for Ubap2l^+/+ MEFs was set to 1. Shown are results for MEFs from 1 Ubap2l^−/− mouse with the standard error of the mean. Representative of 2 independent experiments. (E) Identification of a BMI1 PcG subcomplex containing UBAP2L. HEK 293 cells were transfected with Flag-UBAP2L and cell extracts were fractionated on glycerol gradients. The presence of Flag-UBAP2L, BMI1, RNF2, and PHC1 in the fractions of the gradient was determined by western blotting with the antibodies indicated to the right. *The position of full-length Flag-UBAP2L protein. (F) Model for the role of UBAP2L in the regulation of HSC activity.