Scale pub, 40 m. neuron relating to (C) (mean SEM, 1-way ANOVA, Dunnett post hoc test, = 4 biologically self-employed experiments, approximately 50 cells/experiment normally). The data underlying all the graphs demonstrated in the number are included in S1 Data. CCK-8, cell counting kit-8; DRG, dorsal root ganglion; RSK, ribosomal S6 kinase; SEM, standard error of the mean.(TIF) pbio.3001653.s001.tif (1.9M) GUID:?7B932E53-6B7C-46F7-8722-95096433F36B S2 Fig: RSK1 expression and phosphorylation are up-regulated in DRG following sciatic nerve axotomy. Related to Fig 2. (A) Representative images of in situ hybridization for RSKs in DRG cells sections on days 0 and 4 after nerve injury. The corresponding sense probe was used like a control (Sense) for nonspecific binding. Scale pub, 200 m. (B) Quantification of RSKs intensity relating to (A) (mean SEM, unpaired 2-tailed test, = 4 biologically self-employed animals/group). (C) Western blotting showing RSK1 and RSK2 manifestation in DRG cells after SNI. (D) Quantification of RSK1 and RSK2 manifestation levels relating to (C) (mean TCS ERK 11e (VX-11e) SEM, 1-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). (E) European blotting showing RSK phosphorylation in DRG cells after SNI. (F) Quantification of RSK phosphorylation levels relating to TCS ERK 11e (VX-11e) (E) (mean SEM, 1-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). (G) DRG cells were fractionated into nuclear and cytoplasmic fractions in the indicated time points after SNI. The fractions were immunoblotted for p-RSKS380, p-RSKT573, p-RSKS221, GAPDH (cytoplasmic marker), and Lamin B1 (nuclear marker). (H) Quantification of RSK phosphorylation levels relating to (G) (mean SEM, 2-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). The data underlying all the graphs demonstrated in the number are included in S1 Data. DRG, dorsal root ganglion; RSK, ribosomal S6 kinase; RSK1, ribosomal S6 kinase 1; SEM, standard error of the mean; SNI, sciatic nerve injury.(TIF) pbio.3001653.s002.tif (5.6M) GUID:?9E8FE715-286A-4703-ADF6-8FAD07FA93E7 S3 Fig: RSK1 activities are up-regulated following sciatic nerve axotomy. Related to Fig 2. (A, C) Representative fluorescence images of immunostaining for p-S6S235/236 (A) and p-eEF2K (C) in the DRG on day time 0, 1, or 4 post-SNI. Level pub, 50 m. (B, D) Quantification of p-S6S235/236 (B) and p-eEF2K (D) immunofluorescence intensity relating to (A) and (C) respectively. Relative protein manifestation levels were quantified after normalization to background immunofluorescence (secondary antibody only) (mean SEM, 1-way ANOVA, Dunnett post hoc test, = 5 biologically self-employed animals/group). (E) European blotting showing p-S6S235/236, total S6, p-eEF2K and total eEF2K manifestation in DRG cells post-SNI. (F, G) Quantification of relative p-S6S235/236/S6 (F) and p-eEF2K/eEF2K (G) levels relating to (E) (mean SEM, 1-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). The data underlying all the graphs demonstrated in the number are included in S1 Data. DRG, dorsal root ganglion; RSK1, ribosomal S6 kinase 1; SEM, standard error of the mean; SNI, sciatic nerve TCS ERK 11e (VX-11e) injury.(TIF) pbio.3001653.s003.tif (4.5M) GUID:?FD09FE52-4E8C-43B8-9C79-616FB344DB48 S4 Fig: Determination of efficiency and specificity of shRNAs targeting RSK1 in vitro. Related to Fig 3. (A) RT-qPCR analysis of the manifestation of RSK1 and RSK2 in DRG neurons infected with control AAV2/8 expressing scramble shRNA (Con) or AAV expressing shRNA1 (RSK1-sh1) or RSK1-sh2 (imply SEM, 1-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). (B) Western blotting showing RSK1 and RSK2 manifestation in DRG neurons infected with control AAV2/8 or AAV expressing RSK1-sh1 or RSK1-sh2. TCS ERK 11e (VX-11e) (C) Quantification of RSK1 and RSK2 levels relating to (B) (mean SEM, 1-way ANOVA, Dunnett post hoc test, = 3 biologically self-employed experiments). (D) RT-qPCR analysis of the manifestation of potential candidate target genes of RSK1-sh2 (CACNA1S, EXOC2, PRKCA) in DRG neurons infected with control AAV2/8 or AAV expressing RSK1-sh2 (mean SEM, unpaired 2-tailed test, = 3 biologically self-employed experiments). The data underlying all the graphs demonstrated in the number are included Rabbit polyclonal to ZNF490 in S1 Data. DRG, dorsal root ganglion; RSK1, ribosomal S6 kinase 1; RT-qPCR, reverse transcription quantitative real-time PCR; SEM, standard error of the mean.(TIF) pbio.3001653.s004.tif (1.2M) GUID:?2A3D1304-13A2-4483-9D5B-4EAFFD211FF8 S5 Fig: Determination of AAV2/8 infection efficiency in vivo. Related to Fig 3. (A) EGFP (green) was co-labeled having a neuronal marker NeuN (reddish) in DRG at 2 weeks following intrathecal injection of AAV2/8 expressing EGFP. Level pub, 200 m. (B) Pub graph represents percentage of EGFP-positive neurons in all DRG neurons (mean SEM, = 5 biologically.