Perk1 2 thr202 tyr204 antibody

Cdk5 (Abcam, Cambridge, UK; clone 2G2), cytokeratin 5 (Abcam, EP1601Y), cytokeratin 8 (BioLegend, London, UK; clone 1E8), drebrin (GeneTex, Isleworth, UK; GTX11068), drebrin (Abcam 176318 & 178408), drebrin (Progen, Heidelberg, Germany; clone Mx823), EB1 (BD Transduction Laboratories, Oxford, UK; clone 5), EB3 (Millipore, Consett, UK; AB6033), p35 (Abcam, ab66064), GAPDH (GeneTex, clone GT239), GFP (Abcam, 6556), laminin (Sigma-Aldrich, Poole, UK; clone LAM-89), p35/25 (Cell Signaling, Hitchin, UK; C64B10), p63 (Abcam, ab735 clone BC4A4), pERK1/2 (Thr202/Tyr204) antibody (Cell Signaling; #9101), pS142-drebrin (Millipore, clone 3C14), tyrosinated α-tubulin (SeroTec, Kidlington, UK; clone YL 1/2), vimentin (Dako, Glostrup, Denmark; clone V9). Horseradish peroxidase-conjugated secondary antibodies were from Dako. Alexa-conjugated secondary antibodies and phalloidin were from Life Technologies, Warrington, UK. BTP2 (YM-58483) was from Cambridge Bioscience (Cambridge, UK; CAY13246). Recombinant human CXCL12 was from PeproTech (London, UK; 300-28A). Matrigel was from Corning (New York, USA; 354234) and fibronectin from Millipore.

48 h after lentivirus infection, total protein was extracted from cells by RIPA cell lysis buffer with proteinase and phosphatase inhibitors. The supernatant of lysates was collected and electrophoresed in 10%-12% SDS-PAGE gel, then transferred onto PVDF membranes. Membranes were blocked in Tris-buffered saline plus 0.1% Tween-20 (TBST) with 5% non-fat dry milk or bovine serum albumin (BSA) at room temperature for 1h, followed by primary antibody incubation at 4°C overnight. On the next day after washing with TBST, membranes were incubated with secondary antibodies labeled with HRP at room temperature for 1 h. Then proteins were detected by ECL detection system (P1010, Applygen Technology, Beijing, China). Protein levels were measured by Image J software. Primary antibodies were listed as follows: caspase-3(No. 9662, 1:1000, Cell Signaling Technology), cleaved caspase-3 (No. AF7022, 1:1000, Affinity Biosciences); ERK1/2 antibody (No.4695, 1:1000, Cell Signaling Technology); p-ERK1/2 (Thr202/Tyr204) antibody (No. 4370, 1:1000, Cell Signaling Technology); AKT (No. 9272, 1:1000, Cell Signaling Technology), p-AKT (Ser473) antibody (No. 9271,1:1000, Cell Signaling Technology); cyclinD1 (AB134175, 1:20000,Abcam); β-actin (No. 10494-1-AP, 1:10000, Proteintech, Beijing, China).

Media, sera, antibiotics for cell culture, lipofectamine reagent, Optimem, and Superscript III Reverse Transcriptase were from Invitrogen (Grand Island, NY). Prep1 plasmid cDNA (pRc/CMV-Prep1) has been designed in the laboratory and produced by Invitrogen (Grand Island, NY). The Prep1, BDNF, TrkB, ERK1/2, and 14-3-3θ antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The p-ERK1/2(Thr202/Tyr204) antibody was from Cell Signaling Technology, Inc. (Danvers, MA). Calbidin D-28k antibody was from Swant® (Marly, CH). S-100 antibody was from Ventana Medical System Inc. (Tucson, Arizona, USA). Protein electrophoresis and real-time PCR reagents were purchased from Bio-Rad (Hercules, CA), and Western blotting and ECL reagents from Amersham Biosciences (Arlington Heights, IL). Mayer Hemalum staining was from Bio-Optica S.p.A. (Milan, IT). Tissue-Tek OCT was from Sakura Finetek USA, Inc. (Torrance, CA). Recombinant human/mouse/rat BDNF protein was from Peprotech (Rocky Hill, NJ, USA). Cytochrome C, bovine catalase, DAB, sulforhodamine B, Eukitt, and all other chemicals were from Sigma-Aldrich (St. Louis, MO).