Figure 4.

RBM3 Regulates RTN3 In Vivo

(Ai) Cooling increases RBM3 and RTN3 levels in hippocampi of wild-type mice. Representative western blots are shown. Bar graphs show quantification of RBM3 and RTN3 levels relative to β-actin and GAPDH, respectively. n = 5 control and 5 cooled mice for RBM3. n = 9 control mice and 9 cooled mice for RTN3. ^∗p < 0.05; ^∗∗∗p < 0.001

(Aii) qRT-PCR of RNA isolated from hippocampi of cooled mice showed no significant change in the abundance of RBM3 or RTN3 mRNAs following cooling.

(B) Protein synthesis rates were determined by ³⁵S methionine incorporation into nascent protein using ex vivo hippocampus slices from cooled mice and control mice. A two-tailed paired Student’s t test was used to calculate statistical significance. Error bars represent 1 SD from the mean within three independent experiments. ^∗p < 0.05.

(C) Sucrose density gradient ultracentrifugation performed of cytoplasmic extracts from hippocampi from control and cooled mice. Absorbance plots show the distribution of RNA within subpolysomes (40S, 60S, and 80S) and polysomes.

(D) RTN3 induction on in vivo cooling is dependent on RBM3 protein expression. Knockdown of RBM3 resulted in a 38% decrease in RTN3 induction. n = 6 LV-control and 6 LV-shRNA-RBM3 mice. ^∗∗p < 0.01.