Fig. 2.

Summary of RNA-seq and related analyses of wild-type (wt) vs. 2-KO (hsUBR1^−/− hsUBR2^−/−) human HEK293T cells. (A) IB analyses of the wild-type (parental) HEK293T cell line and two independently produced 2-KO cell lines, #1 and #2, using anti-hsUBR1 and anti-hsUBR2 antibodies. (B) Lanes 1 through 3, fractionated total RNA, stained with ethidium bromide, from wild-type HEK293T cells and 2-KO cell lines #1 and #2. The bands of 18S and 28S rRNAs are indicated on the Left. Lanes 4 through 6, Northern hybridization, using a ³²P-labeled RNA probe specific for the ∼2.1-kb hsADRB2 mRNA, of blotted RNAs in lanes 1 through 3. Lanes 7 and 8, same as lanes 4 and 5, but a 7-fold longer autoradiographic exposure to detect hsADRB2 mRNA in wild-type cells (lane 7). Lanes 9 through 11, same as lanes 4 through 6, but hybridization with a ³²P-labeled RNA probe specific for the ∼1.3-kb hsGAPDH mRNA (loading control). (C) Lanes 1 and 2, IB-based (using anti-hsADRB2 antibody) CHX chase, for 0 and 4 h, of hsADRB2 in wild-type HEK293T cells. Lanes 3 and 4, same as lanes 1 and 2, but with 2-KO cell line #1, in which hsADRB2 mRNA was up-regulated by 17-fold (see B and the main text). (D) The list of 10 human mRNAs that were up-regulated by at least 3.1-fold (from 22-fold to 3.1-fold) in 2-KO cell line #1, and the analogous list of 10 mRNAs that were down-regulated by at least 4.3-fold (from more than 100-fold to 4.3-fold) in the above 2-KO cells. Superscript "a" in the right column denotes a minimal estimate of the extent of observed repression. (E) ³²P-Northern analysis (see B) of the repression of hsMAGE6 mRNA (see D) in 2-KO cell lines #1 and #2 (lanes 2 and 3) vs. robust expression of hsMAGE6 in wild-type cells (lane 1).