Western blotting was conducted as previously described [17 (link)] using anti-HIF-1α (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-cyclin E1 (1:1000; Abcam, Cambridge, United Kingdom), anti-cyclin A2 (1:1000; Abcam), anti-CDK2 (1:1000; Abcam), anti-p21 (1:1000; Abcam), anti-CD99 (1:1000; Abcam), anti-ERK1+ERK2 (1:5000; Abcam), anti-ERK1 (phospho T202)+ERK2 (phospho T185) (1:1000; Abcam) and anti-GAPDH (1:1000; Sangon Biotech Corp., Shanghai, China) primary antibodies. The membranes were then incubated with secondary anti-rabbit antibodies conjugated to horseradish peroxidase (1:4,000; Abcam). Proteins were detected using PierceTM enhanced chemiluminescence (ECL) western blot analysis substrate (Thermo Fisher Scientific Inc.) and analyzed with ImageJ software (NIH, Bethesda, MD, United States).
Anti erk1 erk2
Manufactured by Abcam
Sourced in United Kingdom
Anti-ERK1+ERK2 is a primary antibody that recognizes extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are serine/threonine protein kinases that play a key role in the MAPK/ERK signaling pathway, which is involved in cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Anti p21, by Abcam (1 mentions)
Anti cyclin a2, by Abcam (1 mentions)
Anti cyclin e1, by Abcam (1 mentions)
Anti cd99, by Abcam (1 mentions)
Anti erk1 phospho t202 erk2 phospho t185, by Abcam (1 mentions)
Anti cdk2, by Abcam (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti c myc antibody, by Abcam (1 mentions)
Ecl luminescence reagent, by Thermo Fisher Scientific (1 mentions)
Qpcr mix, by Promega (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein concentration assay kit, by Solarbio (1 mentions)
Protein loading buffer, by Solarbio (1 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Santa Cruz Biotechnology (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
4 protocols using anti erk1 erk2
1
Western Blotting Procedure for Protein Analysis
2
Lipofectamine 3000 Transfection and Analysis
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Lipofectamine™ 3000 transfection reagent was obtained from Invitrogen (Carlsbad, USA). Cell counting kit-8 (CCK-8) was provided by Dojindo (Kumamoto, Japan). anti-ERK1ERK2 (phospho T202/Y204, SP327), anti-c-Myc (phospho S62) antibody, anti-MNK1 (phospho T385, EPR2370), anti-ERK1+ ERK2, anti-MNK1 and anti-c-Myc antibodies were obtained from Abcam (London, United Kingdom). PTPRF/LAR (E6W4X) rabbit mAb was obtained from Cell Signaling Technology (Massachusetts, United States). BCA protein concentration assay kit and 5× protein loading buffer were provided by Beijing Solarbio life sciences(Beijing, China). NucleoZol was obtained from Gene Co., Ltd (Hongkong, China). ECL luminescence reagent was obtained from Thermo Scientific (Waltham, USA). qPCR Mix was provided by PROMEGA (Madison, MA, United States).
3
Western Blot Analysis of MC3T3-E1 Cells
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MC3T3-E1 cells were lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1% phosphatase inhibitor cocktail [Nacalai Tesque, Kyoto, Japan]). The cell lysates were separated by SDS-polyacrylamide gel electrophoresis and then were electrophoretically transferred to PVDF membranes (Bio-Rad, MA, USA). For immunodetection, the following antibodies were used: anti-β-tubulin (Sigma-Aldrich), anti-Akt (Cell Signaling Technology), anti-phospho-Akt (Ser473; Cell Signaling Technology), anti-ERK1+ERK2 (Abcam Inc., Boston, USA) and anti-phospho-ERK (Tyr 204: Santa Cruz, CA, USA). The Envision+/HRP system (Dako, Glostrup, Denmark) was used and protein bands were visualized by 0.02% 3,3’-diaminobenzidine (Dohjin Laboratories, Kumamoto, Japan). The relative densities of each band against that of β-tubulin on monochrome photographs were determined with Image J software (Image J 1.45, NIH, USA).
4
Hippocampal Protein Expression Analysis
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The total proteins of hippocampal tissues and primary neurons were isolated using radioimmunoprecipitation assay buffer (RIPA). The protein concentration of all tissue lysates was determined with a bicinchoninic acid assay (BCA) protein assay kit. Same amount of total protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, Unite States). The membranes were blocked with 5% non-fat powdered milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature (RT) and then incubated overnight at 4°C with the appropriate primary antibodies: anti-Arc, anti-PKA (phospho T197; p-PKA), anti-ERK1 + ERK2 (phospho T202 + T204; p-ERK1/2), and anti-CREB (phospho S133; p-CREB) (Abcam, Cambridge, MA, Unite States). After washing in TBST, the membrane was incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, and protein bands were visualized using the Immun-StarTM HRP Chemiluminescence Kit (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal loading control.
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