MBP and sncRNA715 Expression in Schwann cells.

<p><b>A</b>, Reverse transcription PCR (RT-PCR) on RNA extracted from Oli-<i>neu</i> or IMS32 cells using <i>Mbp</i>-specific primers. The 88nt long amplicon for <i>Mbp</i> was visualized in an ethidium bromide-stained 4% agarose gel. <b>B</b>, Western Blots of lysates from P18 mouse brain (brain lysate), primary oligodendrocytes (pOL, 7DIV), IMS32 and Oli-<i>neu</i> cells using MBP and GAPDH (loading control) specific antibodies. <b>C</b>, Reverse transcription PCR (RT-PCR) on RNA extracted from Oli-<i>neu</i> or IMS32 cells using a sncRNA715-specific primer assays. PCR products (~60-nt long due to the use of hairpin primers in the RT reaction) were visualized in an ethidium bromide stained 4% agarose gel. <b>D</b>, Northern Blots with RNA from IMS32 and undifferentiated primary Schwann cells (pSC) shows expression of sncRNA715 in IMS32 and a lower expression in pSC. Synthetic sncRNA715 (715-mimic) and U6 snRNA were used as positive control and loading control, respectively. <b>E</b>, RT-PCR on RNA from IMS32 and undifferentiated pSC confirms lower expression of sncRNA715 in pSC compared to IMS32 cells shown in D. 715-mimic was used as positive control and snoRNA135 as loading control.</p

Inverse correlation of MBP and sncRNA715 in the sciatic nerve.

<p><b>A&B</b>, The sciatic nerve was lysed from mice at postnatal day 1, 4 and 9 and myelin proteins as well as sncRNA715 expression was analyzed by Western blotting (<b>A</b>) and qPCR (<b>B</b>), respectively. MBP and CNP Western blots show increasing levels in differentiating sciatic nerves (<b>A</b>) while sncRNA715 levels decrease during differentiation, P-values P4: 0,0313, P9: 0,0313 (<b>B</b>, log2 values are plotted, sncRNA715 levels at P4 and P9 were quantified relative to P1 using snoRNA135 as a reference gene). Number of experiments (n) are indicated and bar graphs represent mean values ± s.e.m. (Wilcoxon signed-rank test, *P< 0.05, GraphPad Prism5 was used for statistical analysis).</p

Inverse correlation of MBP and sncRNA715 in primary Schwann cells.

<p><b>A</b>, Primary Schwann cells derived from sciatic nerves of P3 Wistar rats were cultured in non-differentiating (untreated) or differentiating (+NRG1 +dbcAMP) conditions. MBP protein can only be detected by immunocytochemistry in differentiated Schwann cells. Scale bar represents 50μm. <b>B</b>, Western Blots of undifferentiated and differentiated primary Schwann cells show MBP protein only present in differentiated Schwann cells. CNP is expressed in both maturation stages of primary Schwann cells. GAPDH serves as loading control. <b>C</b>, MBP and sncRNA715-specific RT-PCR on RNA extracted from undifferentiated or differentiated primary Schwann cells. <i>Mbp</i> mRNA is present at both differentiation states while sncRNA715 is detectable in undifferentiated and hardly in differentiated Schwann cells. SnoRNA135 and <i>G6pdh</i> mRNA were used as loading controls.</p