Figure 3.
lnc-31 Interacts and Stabilizes YB-1 Protein that Controls Rock1 Translation
(A) Western blot analysis of proteins retrieved from lnc-31 RNA pull-down.
(B) Upper: western blot with YB-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (negative control) antibodies of protein extracts from YB-1 CLIP experiment. Input sample (IN) accounts for 2% of the extract. Lower: graph showing the results of two CLIP experiments using YB-1 antibodies. The graph shows the enrichment (%IN) in IP and IgG samples for Yb-1, Akt1, lnc-31, and Rock1 transcripts and for the control reference Sngh12 RNA.
(C) Western blot of proteins from C2C12 cells treated with SCR and si-YB-1 using YB-1 and ROCK1 antibodies. ACT is used as loading control. Quantification of ROCK1 protein levels from three independent experiments is shown below. ROCK1 levels were normalized against ACT levels and expressed with respect to the SCR sample set to a value of 1.
(D) Graph showing the results of two CLIP experiments using YB-1 antibodies and C2C12 cells treated with SCR and si1-lnc-31. The graph shows the enrichment (%IN) in IP and IgG samples for the Yb-1 and Rock1 mRNAs.
(E) Western blot analysis using YB-1 antibodies on proteins from C2C12 cells treated with SCR or si1-/si2-lnc-31 or transfected with either pcDNA3.1 (CTRL) or plnc-31. ACT was used as loading control. Densitometric analyses of three independent experiments are shown below.
(F) Upper: western blot of proteins from C2C12 cells treated with CHX in the presence of SCR or si1-lnc-31 for the indicated time. Lower: densitrometric analyses of YB-1 protein levels from three independent experiments. YB-1 protein levels were normalized against ACTIN levels and expressed with respect to the SCR and si-lnc-31 time “0” set to a value of 1.
(G) Western blot analysis, using YB-1 and CDKN1B antibodies, of proteins from C2C12 cells treated as in (D) (left) or with si1-lnc-31 in the presence of DMSO or MG132 (right). ACT was used as loading control. Densitometric analyses of three independent experiments are shown below.
Error bars represent SD of three independent experiments (unless differently specified). ∗p < 0.05 and ∗∗p < 0.01 correspond to paired two-tailed Student’s t tests.