G lisa assay

GTP-bound RhoA was assessed with a G-LISA assay (Cytoskeleton, Inc. Denver, CO, USA). For in vivo tissue samples, spinal cord tissue was excised from the spinal cord at 3 days post injury and 1 cm of spinal cord tissue centered at the injury epicenter was flash frozen in liquid nitrogen. For in vitro Rho activity assessment, tumor necrosis factor-α (TNFα) (40 ng/ml) + IFNγ (50 ng/ml) treated M1 microglia were grown on PDL or PDL + CSPGs with and without ILP (10 μM) and ISP (10 μM) for 1 day then harvested on ice and flash frozen in liquid nitrogen. For G-LISA assessment, 50 μg of protein was used for both in vivo and in vitro assessments. G-LISA was performed as per manufacturer’s instructions.

Protein was extracted from MLO‐A5 cultures and RhoA activation measured by the GLISA assay (Cytoskeleton, Denver), which specifically recognises active GTP‐bound RhoA. The quantity of GTP‐bound RhoA was then measured by luminometry and expressed in relative light units (RLU).

The analysis was done by G-LISA assay (Cytoskeleton Inc., Denver, USA) according to the manufacturers protocol. In brief, LNT229 (Neo-control or sCPE-overexpressing) or LN18 (si-mock or si-CPE) cells were cultured under standard conditions until the cells reached 40% confluence and then under serum-starving conditions for 24h. The cells were stimulates with 1% FCS in DMEM for 30 min and harvested on ice followed by snap freezing in the liquid nitrogen. For the assay test, either unstimulated (under serum-reduced conditions) or stimulated with 50 ng/ml EGF U87 GBM cell line was taken, with agreement to the protocol recommendations for Rac1 activation. Lysis buffer was used as blank and unhydrolyzed Rac1 protein was supplied with the kit as a positive control. The colorimetric signal was measured at 490 nm.

Cells or tissues were lysed in 100 μl of loading buffer (2% sodium dodecyl sulfate, 30% glycerol, 300 mM β-mercaptoethanol, 100 mM Tris-HCl pH 6.8): extracts were separated on SDS-10% polyacrylamide gels, transferred and incubated with the relative antibodies and developed according to the manufacturer’s instructions (GeneSpin). An anti-ArhGAP15 monoclonal antibody was also used, this was raised in mouse against a GST-fusion protein of an ArhGAP15 peptide spanning from aminoacids 220–320. For loading control, an anti-actin mouse monoclonal antibody was used (from Sigma).
Rac1/Rac3 activity was measured by pull-down assay, as described18 (link). Detailed protocol in Supplementary Methods. The clone 23A8 mouse anti-Rac1 antibody was used (Upstate Biotech, used 1:2000) which, however, also recognizes Rac3 in Western blot analyses (data not shown). The amount of GTP-bound Rac1/Rac3 was also determined with the G-LISA assay (Cytoskeleton, Inc Denver, CO, USA), as previously described69 (link). This assay has not been tested for specificity against Rac3.

For assaying Rac GTPase activity, G-LISA assay (Cytoskeleton Inc., Denver, CO, USA) was performed according to the manufacturer’s instructions.