Western blotting was performed as described previously [39 (link)–41 (link)]. The hippocampus was collected from deeply anesthetized rats (n = 8 per group, 40 mg/kg, i.p.) and snap-frozen in liquid nitrogen. Tissues were homogenized on ice in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma–Aldrich). After centrifugation, the supernatants were collected and denatured with SDS–PAGE loading buffer for 5 min at 95°C. Equal amounts of protein were separated by SDS–PAGE and transferred to PVDF membranes (GE Healthcare Life Science). After blocking with 5% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C (rabbit anti-pCREB, 1 : 1000, rabbit anti-CREB, 1 : 1000, Cell Signaling Technology; rabbit anti-BDNF, 1 : 600, Abcam). Then, the membranes were washed with TBST and incubated with secondary antibodies (1 : 1000, Santa Cruz Biotechnology) for 1 h at room temperature. Finally, bands were detected with an enhanced chemiluminescence reagent eECL Kit (CWBio, China) and quantified using ImageJ software (NIH, USA). GAPDH (rabbit antibody, 1 : 5000, Abcam) was used as the loading control.
Eecl kit
Manufactured by CWBIO
Sourced in China
The EECL Kit is a laboratory equipment product designed for performing Enzyme-Enriched Cell Lysis. It provides the necessary components and protocols for efficient cell lysis and extraction of cellular contents, supporting various downstream applications in life science research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti creb, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Gapdh, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti pcreb, by Cell Signaling Technology (1 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
Lysis buffer, by CWBIO (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Superoxide dismutase sod assay kit, by Nanjing Jiancheng (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
2 nbdg, by Thermo Fisher Scientific (1 mentions)
Antibiotics 100 penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by CWBIO (1 mentions)
Donkey anti rabbit igg h l alexa fluor 647, by Abcam (1 mentions)
Malondialdehyde mda, by Nanjing Jiancheng (1 mentions)
3 protocols using eecl kit
1
Quantitative Analysis of Hippocampal Signaling Pathways
2
Western Blot Analysis of IRAK1, TRAF6, and pNF-κB in Rat Dorsal Root Ganglia
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Total proteins from rat L4-L6 DRGs or SDH were extracted with lysis buffer (CWBio, Beijing, China). Briefly, 30 μg of each sample was resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto Immobilon-P polyvinylidene difluoride (GE). After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with an anti-IRAK1 antibody, anti-TRAF6 antibody, anti-pNF-κB (p65) antibody, and anti-β-actin antibody. The corresponding secondary antibodies were probed after washing the membranes. Final results were acquired using a western blot detection system (GE) with enhanced chemiluminescence reagents eECL Kit (CWBio, Beijing, China). Table 3 lists the primary and secondary antibodies used for the western blot analysis.
List of primary and secondary antibodies used for western blot analysis
| Antibody | Host | Company | Catalog number | Dilution | Incubation conditions |
|---|---|---|---|---|---|
| IRAK1 | Rabbit | Abcam | ab238 | 1:1000 | Overnight 4 °C |
| TRAF6 | Rabbit | Abcam | ab181622 | 1:1000 | Overnight 4 °C |
| pNF-κB (p65) | Mouse | Cell signaling technology | #13346 | 1:1000 | Overnight 4 °C |
| β-actin | Mouse | ZSGB-BIO | TA-09 | 1:1000 | Overnight 4 °C |
| Anti-rabbit IgG horseradish peroxidase (HRP) | Goat | ZSGB-BIO | ZDR-5306 | 1:3000 | 1 h RT |
| Anti-mouse IgG horseradish peroxidase (HRP) | Goat | ZSGB-BIO | ZDR-5307 | 1:3000 | 1 h RT |
3
Cellular Signaling Pathway Analysis
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GF2 (purity,≥99%) was purchased from Chengdu PhytoElite Biotech (Chengdu, China). The MEM medium was obtained from Hyclone (Logan, UT, USA). Antibiotics (100 × penicillin/streptomycin) and Trypsin-EDTA were purchased from Gibco (California, USA). Fetal bovine serum (FBS) was purchased from Corning (New Zealand). 2-NBDG and TRIzol reagent were from Invitrogen (Carlsbad, CA, USA). cDNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FastStart Universal SYBR Green Master was provided from Roche (Mannheim, Germany). Malondialdehyde (MDA) and superoxide dismutase (SOD) assay kits were acquired form Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The kits for reactive oxygen species assay, glycogen PAS stain and glycogen content assay were purchased from Solarbio (Beijing, China). Cell lysis buffer and eECL kit were purchased from CWBio (Jiangsu, China). The primary antibodies against p-JNK (#4671), JNK (#9258), p-ERK1/2 (#8544), ERK1/2 (#4348), p-p38 MAPK (#4511), p38MAPK (#9212), NF-κB p65 (#8242), p-AKT (#4060), AKT (#9272), p-PDK1 (#3438), PDK1 (#3062), p-GSK-3β (#5558), GSK3β (#12456), Anti-β-actin pAb-HRP-DirecT (#PM053-7) was from Medical & Biological Laboratories CO., LTD (Nagoya, Japan). Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150075) was from Abcam (Cambridge, MA, USA).
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