Anti gap43

Control and treated cells were given a washing with chilled 1X PBS followed by fixation with acetone and methanol (1:1) and permeabilization with 0.3% Triton- X100 in 1X PBS. Cells were then blocked with 2% BSA and incubated with primary mouse monoclonal antibody anti-α-Tubulin (1:500), anti-NF-κB (1:500), anti-MAP-2 (1:250), anti-NF200 (1:500), anti-GAP 43 (1:250), anti-HSP70 (1:500), anti-Mortalin (1:500), anti-Bcl-xL (1:200), anti-Cyclin D1(1:250), anti-NCAM (1:250), rabbit monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich) and mouse monoclonal anti-PCNA (1:250), mouse polyclonal anti-PSA-NCAM (1:250) (from Millipore, MA, USA) for 24 h in humid chamber at 4 °C. No permeabilization was carried out for PSA-NCAM immunostaining. After primary antibody incubation, three washings were given with 0.1% PBST and incubated with secondary antibody (goat anti-mouse/ rabbit IgG/ IgM Alexa Fluor 488/543) for 2 h at RT. Cells were stained with nuclear staining dye DAPI (Sigma-Aldrich) for 15 min, washed with 0.1% PBST and mounted with antifading agent Fluoromount (Sigma-Aldrich). Images were captured with Nikon AIR Confocal Laser Scanning Microscope and analyzed with NIS elements analysis software version 4.11.00 (Nikon Co., Tokyo, Japan). Each experiment was carried out in triplicate.

Cells were harvested and lysed respectively in IP Lysis Buffer (Thermo, USA) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). The protein samples were incubated with indicated antibody in 1 mL IP Lysis Buffer overnight at 4 °C, and then were precipitated with 20 μL Protein A/G Plus-agarose (Roche, Switzerland). After a brief centrifugation, the pellet was washed 3 times with IP Lysis Buffer. The lysates and immunoprecipitates were analyzed by immunoblotting.
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .

The following antibodies were used in this study: Affinity-purified neuroserpin apK53, a rabbit polyclonal antibody raised to a synthetic peptide corresponding to the 19 C-terminal amino acids of rat neuroserpin [20] , anti-FLAG (Cat No: F3165, Sigma-Aldrich Co. LLC, MO, USA), anti-GAPDH (Cat No: ab8245, Abcam, Cambridge, UK), anti-synapsin (Cat No: 611392, BD Biosciences, CA, USA), anti-PSD-95 (Cat No: MAI-045, Thermo Scientific, IL, USA), anti-NR1 (Cat No: 556308, BD Biosciences, CA, USA), anti-Tau (Cat No: MAB3420, Millipore, MA, USA), anti-GAP-43 (Cat No: G9264, Sigma-Aldrich Co. LLC, MO, USA), goat anti-rabbit IgG (H+L) Horseradish Peroxidase-conjugated antibody (Cat No: 111-035-003, Jackson ImmunoResearch Laboratories, Inc., PA, USA), and goat anti-mouse IgG (H+L) Horseradish Peroxidase-conjugated (Cat No: 115-035-003, Jackson ImmunoResearch Laboratories, Inc., PA, USA).