Supplementary figure 1 (A) Quantitative analysis of F-actin signal intensity in NIH3T3 cells treated with PTD4-myc- RBD. NIH3T3 cells were treated with PTD4-myc-RBD as described. Please note the increase in the F-actin signal intensity under the cell membrane (left) which is in correlation with the increase of myc intensity (Fig.1E). The F-actin intensity remained unchanged in the intermediate zone among the stress fibers as comparing to serum untreated cells. (B) p75-fc chimera blocks the activation of Rho after Nogo addition. Cerebellar granule neurons were treated with PTD4-myc-RBD as described, and 2 hours before Nogo
addition 10µg/ml of p75-fc chimera was added into the medium. The middle panel shows diffuse distribution of PTD4-myc-RBD without any colocalization with p75 NTR signal. The result is consistent with the notion that p75-fc blocks activation of Rho by myelin-derived products. Scale bar = 10 µm. (C) Activation of Rho in CGN plated on myelin. CGN were plated on myelin and the assay was performed as described. The signal for PTD4-myc- RBD in the middle panel shows colocalization with the p75 NTR signal (left) panel. Scale bar = 10 µm.
Supplemetary Figure 2 Colocalization of myc staining with anti-neuron-specific b tubulin III (TuJ-1) in the spinal cord. The images are taken from corticospinal tract 3 mm from the injury site in the rostral stump. PTD4-myc-RBD was administered via the tail vein of the rats with spinal cord injury. Note the colocalization of myc with TuJ1, showing that the fibers are neurons. Scale bar = 20 µm.
Supplemetary Figure 3
PTD4-myc-RalGDS does not induce cytoskeletal changes in the NIH3T3 cells. (A) Structure of PTD4-myc-RalGDS. RalGDS-Rap binding domain of RalGDS. (B) Purification of PTD4-myc-RalGDS. PTD4-myc-RalGDS was produced as a GST-fused protein, and the GST moiety was removed. Coomasie blue staining (left) and immunoblot with the anti-myc antibody (right) are shown. (C) PTD4-myc-RalGDS does not detect the active form of Rho. Serum starved NIH 3T3 cells were either treated or not treated with the serum. The cells were stained with anti-myc antibody (MYC) and phalloidin (F-actin). The PTD4-myc-RalGDS signal is not colocalized with F-actin and shows no detectable changes in the distribution after 10% FBS stimulation. Ral, PTD4-myc-RalGDS. Scale bar = 10 µm. (D) Quantitative analysis of PTD4-myc-RalGDS and F-actin signal distribution following serum stimulation. Note that no significant changes of myc signal intensity were observed after serum stimulation. PTD4-myc-RalGDS signal intensity does not correlate with the increase in signal intensity of F-actin under the cell membrane. The total intensity of myc signal was not affected by serum stimulation.
Supplementary Figure 4 PTD4-myc-20aa does not induce cytoskeletal changes in the serum starved NIH3T3 cells after fetal bovine serum addition. (A) Structure of PTD4-myc-20aa. 20aa - 20 random amino acids with the sequence NSRAGYAGRTQSCRGNGIRM. (B) Purification of PTD4- myc-20aa. PTD4-myc-20aa was produced as a GST-fused protein, and the GST moiety
was removed. Coomasie blue staining (left) and immunoblot with the anti-myc antibody (right) are shown. (C) PTD4-myc-20aa does not detect the active form of Rho. Serum starved NIH 3T3 cells were either treated or not treated with the serum. The cells were stained with anti-myc antibody (MYC) and phalloidin (F-actin). The PTD4-myc-20 signal is not colocalized with F-actin (Fig.1D) and shows no detectable changes in the distribution after 10% FBS addition. 20 aa, PTD4-myc-20aa. Scale bar = 10 µm. (D) Quantitative analysis of PTD4-myc-20aa and F-actin signal intensity following serum stimulation. Note that no significant changes of myc signal intensity were observed after serum stimulation. PTD4-myc-20aa signal intensity does not correlate with the increase in signal intensity of F-actin under the cell membrane. The total intensity of myc signal was not affected by serum stimulation.
Supplementary Figure 5 Scrambled PTD4-myc-RBD is not incorporated into the NIH3T3 cells. (A) Structure of ScrPTD4-myc-RBD. ScrPTD4 scrambled version of PTD4 with the amino acid sequence; ARARARYAQAA. (B) Purification of ScrPTD4-myc-RBD. ScrPTD4-myc-RBD was produced as a GST-fused protein, and the GST moiety was removed. Coomasie blue staining (left) and immunoblot with the anti-myc antibody (right) are shown. (C) ScrPTD4- myc-rbd is not incorporated into the cells. Serum starved NIH 3T3 cells were either treated or not treated with the serum. The cells were stained with anti-myc antibody (MYC) and phalloidin (F-actin). No signal was detected for ScrPTD4-myc-RBD in the cells. Scr = ScrPTD4-myc-RBD. Scale bar = 10 µm.
Supplementary Figure 6 PTD4-myc-RalGDS does not detect Rho activation in CGN after Nogo addition. Cerebellar granule neurons were incubated with PTD4-myc-RalGDS (1 µm) and either treated or not treated with the Nogo peptide (10 µm) for 10 min. Immunostaining was carried out with the anti-myc antibody and the anti-p75 antibody. Representative single optical sections for p75 NTR (left), PTD4-myc-RalGDS (middle) and overlay images (right) are shown. Note the weak and diffuse myc signal in both, Nogo treated and untreated CGN, showing that PTD4-myc-RalGDS was unable to detect Rho activation in the cells. Ral = PTD4-myc-RalGDS. Scale bar = 10 µm.
Supplementary Figure 7 PTD4-myc-20aa does not detect Rho activation in CGN after the Nogo peptide addition. Cerebellar granule neurons were incubated with PTD4-myc-20aa (1 µm), and either treated or not treated with the Nogo peptide (10 µm) for 10 min. Immunostaining was carried out with the anti-myc antibody and the anti-p75 antibody. Representative single optical sections for p75 NTR (left), PTD4-myc-RalGDS (middle) and overlay images (right) are shown. Note the diffuse myc signal in both, Nogo treated and untreated CGN, showing that PTD4-myc-20aa was unable to detect Rho activation in the cells. 20aa, PTD4-myc 20aa. Scale bar = 10 µm.
Supplementary Figure 8 ScrPTD4-myc-RBD is not incorporated into the cerebellar granule neurons. Cerebellar granule neurons were incubated with ScrPTD4-myc-RBD (1 µm) and either treated or not treated with the Nogo peptide (10 µm) for 10 min. Immunostaining was carried out with the anti-myc antibody and the anti-p75 antibody. Absence of myc signal demonstrates that ScrPTD4-myc-RBD was not incorporated into the cells. Scr, ScrPTD4-myc-RBD. Scale bar = 10 µm.
Supplementary Figure 9
PTD4-myc-RalGDS does not detect the Rho activation after spinal chord injury. (A) Representative longitudinal sections of spinal cord of injured and sham-operated animals, which received PTD4-myc-RalGDS intravenously. Images of the white matter were taken from the rostral stump from the area of corticospinal tract 3 mm from the transection site and the corresponding site in sham-operated rats. Images of the grey matter were taken from the base of the dorsal horn 3 mm rostrally from the transection site. The sections of injured and sham-operated spinal cord were immunostained with the anti-myc antibody and the anti-p75 antibody. p75 NTR immunoreactivity is induced in the fibers in the white matter after the injury as well as in the neurons in the grey matter. There is no detectable difference in the myc staining in both white and grey matter. Ral+, PTD4-myc-RalGDS administered; RBD, PTD4-myc-RalGDS not administered. Scale bar = 20 µm. (B) Quantitative analysis of PTD4-myc-RalGDS signal intensity in the white matter in the injured and the sham-operated (intact) animals. There was no significant difference in the optical intensity of the myc signal between the injured and the sham-operated animals. The data is mean ± SEM. Range of optical intensity 0 255.
Supplementary Figure 10
PTD4-myc-20aa does not detect the Rho activation after spinal chord injury. (A) Representative longitudinal sections of spinal chord of injured and sham-operated animals which received PTD4-myc-20aa intravenously. Images of the white matter were taken from the rostral stump from the area of corticospinal tract 3 mm from the transection site and the corresponding site in sham-operated rats. Images of the grey matter were taken from the base of the dorsal horn 3 mm rostrally from the transection site. The sections of injured and sham-operated spinal cord were immunostained with the anti-myc antibody and the anti-p75 antibody. p75 NTR immunoreactivity is induced in the fibers in the white matter after the injury as well as in the neurons in the grey matter. Their is no detectable difference in the myc staining in both white and grey matter. 20aa+ = PTD4-myc-20aa administered, 20aa- = PTD4-myc-20aa not administered. Scale bar = 20 µm. (B) Quantitative analysis of PTD4-myc-RalGDS signal intensity in the white matter in the injured and the sham-operated (intact) animals. There was no significant difference in the optical intensity of the myc signal between the injured and the sham-operated animals. The data is mean ± SEM. Range of optical intensity 0 255.
Supplementary Figure 11
ScrPTD4-myc-RBD does not cross the blood brain barrier. Representative longitudinal sections of spinal chord of injured and sham-operated animals which received ScrPTD4- myc-rbd intravenously. Images of the white matter were taken from the rostral stump from the area of corticospinal tract 3 mm from the transection site and the corresponding site in sham-operated rats. Images of the grey matter were taken from the base of the dorsal horn 3 mm rostrally from the transection site. The sections of injured and shamoperated spinal cord were immunostained with the anti-myc antibody and the anti-p75 antibody. p75 NTR immunoreactivity is induced in the fibers in the white matter after the injury as well as in the neurons in the grey matter. There is no detectable signal in the myc staining in both white and grey matter. Scr+ = ScrPTD4-myc-RBD administered, Scr- = ScrPTD4-myc-RBD not administered. Scale bar = 20 µm.