Horseradish peroxidase hrp conjugated secondary antibody

Proteins were collected from cultured cells in Radio Immuno Precipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China). The concentrations of proteins were detected via BCA protein kit (Beyotime). Aliquots of protein were separated by 12% SDS-PAGE and resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% milk in PBS with 0.1% Triton X-100 and incubated with different primary antibodies: anti-RACK1 antibody (ab62735, 1:1,000, 30 kDa, Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (ab15148, 1:500, 135 kDa, Abcam, USA), anti-N-cadherin antibody (ab18203, 1:1,000, 125 kDa, Abcam), anti-Snail antibody (ab82846, 1:500, 68 kDa, Abcam), anti-phosphorylation (p)-β-catenin antibody (ab27798, 1:500, 86 kDa, Abcam), anti-β-catenin antibody (ab32572, 1:5,000, 92 kDa, Abcam), anti-c-Jun antibody (ab32137, 1:1,000, 43 kDa, Abcam) and anti-GAPDH (ab181602, 1:10,000, 36 kDa, Abcam, USA) overnight at 4°C. The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech, Rosemont, IL, USA). Protein bands were detected with enhanced chemiluminescence (ECL, Thermo Fisher Scientific) and visualized by Quantity one (Bio-Rad Laboratories Inc., Hercules, CA, USA).

For protein extraction, RIPA buffer was used to lyse cells and tissue (10 mm of the spinal cord containing the injury epicenter). Protein concentrations were detected by using a Bicinchoninic acid (BCA) assay kit. The denatured proteins were electrophoretically transferred to a 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated at 4°C overnight with a mouse anti-acetylated tubulin monoclonal antibody (1:1,000; Proteintech, USA), rabbit anti-H3K27me3 antibody (1:1,000; CST, USA), rabbit anti-NeuroD1 antibody (1:1,000; Abcam, USA), rabbit anti-UTX antibody (1:1,000; Abcam, USA), actin (1:5,000; Abcam, USA), rabbit anti-β-tubulin antibody (1:1,000; CST, USA), and anti-GAPDH antibody (1:5,000; Abcam, USA). After the membranes were washed with 1% Tris-HCl+Tween (TBST) 3 times (15 min each), they were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; Proteintech, USA) for 90 min. Immunoreactive bands were visualized using enhanced chemiluminescence.

Total proteins of cells and tissues were extracted by lysing in radioimmunoprecipitation assay buffer (RIPA buffer; Beyotime Institute of Biotechnology, Jiangsu, China) on ice for 30 min and then centrifuged at 17,000 × g for 40 min at 4°C. The concentration of protein was determined by a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). An equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Tween 20 with Tris-buffered saline (TTBS) with 10% non-fat milk was used to block the membranes at room temperature for 2 h. Then, membranes were incubated in primary antibody of EIF4A3 (1:500, Proteintech, Rosemont, IL, USA), EGR3 (1:300, Santa Cruz Biotechnology, Santa Cruz, TX, USA), PKP2 (1:200, Santa Cruz Biotechnology, Santa Cruz, TX, USA), PI3K, Akt (1:500, Proteintech, Rosemont, IL, USA), and GAPDH (1:5,000, Proteintech, Rosemont, IL, USA) overnight at 4°C followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Proteintech, Rosemont, IL, USA) at room temperature for 2 h. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology) and scanned by ChemImager 5500 v2.03 software.

IL was purchased from the National Institute for Food and Drug Control (Beijing, China). MTT was purchased from BioFroxx (Darmstadt, Germany). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, USA). Crystal violet was purchased from Solarbio (Beijing, China). Hoechst 33258 was purchased from Sigma-Aldrich (St Louis, USA). DME/F12 medium and RPMI 1640 medium were purchased from HyClone Laboratories (Logan, USA). GSH and oxidized glutathione disulfide (GSSG) assay kit was purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies against Bax (#50599-2-lg, 1:1000), p53 (#10442-1-AP, 1:1000), GAPDH (#10494-1-AP, 1:6500), PARP (#13371-1-AP, 1:500), and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from ProteinTech Group (Rosemont, USA). Primary antibodies against Akt (#9272, 1:2000), anti-phospho-Akt (Ser473) (#9271, 1:2000), Bcl-2 (#2876, 1:1000) and caspase-3 (#9662, 1:1000) were purchased from Cell Signaling Technology (Danvers, USA). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from CoWin Biosciences (Beijing, China).