Each human RCC cell line was plated at a density of 25 000 cells/well on a 24‐well cell culture plate and cultured for 24 h. After removing the medium, cellular proteins were extracted with RIPA buffer (Thermo Scientific, Waltham, MA, USA) and separated by SDS‐polyacrylamide gel electrophoresis. Anti‐β‐catenin antibody (Abcam, Cambridge, UK) and anti‐phosphorylated ERK antibody (Cell Signaling Technology, Beverly, MA, USA) were used as primary antibodies. Anti‐SULF‐2 (H2.1) antibody was produced by MBL as described in the above section. Antibody‐bound protein bands were visualized using ECL Advance Western detection reagents (GE Healthcare, Buckinghamshire, UK) and imaged using a ChemiDoc XRS plus system (BIO‐RAD, Hercules, CA, USA). Individual bands were quantified with Image Lab 2.0 software (BIO‐RAD), and normalized against the β‐actin band. Experiments were conducted at least three times.
Anti phosphorylated erk antibody
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
The Anti-phosphorylated ERK antibody is a laboratory tool designed to detect and study the phosphorylation state of extracellular signal-regulated kinase (ERK) proteins. It is a specific and sensitive reagent that can be used in various immunoassay techniques to identify and quantify the levels of activated, phosphorylated ERK in biological samples.
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Lab products found in correlation
Anti erk antibody, by Cell Signaling Technology (2 mentions)
Anti phosphorylated akt antibody, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Image lab 2, by Bio-Rad (1 mentions)
Anti β catenin antibody, by Abcam (1 mentions)
Ecl advance western detection reagents, by GE Healthcare (1 mentions)
Chemidoc xrs plus system, by Bio-Rad (1 mentions)
Panobinostat, by Cayman Chemical (1 mentions)
Anti jnk antibody, by Cell Signaling Technology (1 mentions)
Anti stat3 antibody, by Cell Signaling Technology (1 mentions)
Anti p65 antibody, by Cell Signaling Technology (1 mentions)
Anti p38 antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions)
Anti pi3k antibody, by Cell Signaling Technology (1 mentions)
Anti phosphorylated p38 antibody, by Cell Signaling Technology (1 mentions)
Anti acetyl histone h4 antibody, by Merck Group (1 mentions)
Anti phosphorylated stat3 antibody, by Cell Signaling Technology (1 mentions)
Rhigf1, by R&D Systems (1 mentions)
Ecl detection kit, by Bio-Rad (1 mentions)
Anti akt pan antibody, by Cell Signaling Technology (1 mentions)
4 protocols using anti phosphorylated erk antibody
1
Western Blot Analysis of RCC Cell Lines
2
Signaling Pathway Regulation in Cell Lines
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The following reagents were purchased from the indicated manufacturers: rabbit polyclonal anti-human DR4 antibody, anti-human TRPV1 antibody, and anti-human HDAC1 antibody from Abcam (Cambridge, UK); mouse polyclonal anti-β-actin antibody and valproate from Sigma (St. Louis, MO); rabbit polyclonal anti-Histone H3 antibody, rabbit polyclonal anti-acetyl-histone H3 antibody and anti-acetyl-histone H4 antibody from Merck Millipore (Billerica, MA); rabbit polyclonal anti-Sp1 antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-PI3K antibody, anti-phosphorylated PI3K antibody, anti-STAT3 antibody, anti-phosphorylated STAT3 antibody, anti-p38 antibody, anti-phosphorylated p38 antibody, anti-p65 antibody, anti-phosphorylated p65 antibody, anti-ERK antibody, anti-phosphorylated ERK antibody, anti-JNK antibody, anti-phosphorylated JNK antibody, horseradish peroxidase (HRP)-anti-rabbit IgG and anti mouse IgG from Cell Signaling Technology (Beverly, MA); rh IGF-1 from R&D Systems (Minneapolis, MN); panobinostat from Cayman Chemical Company (Ann Arbor, MI); and 5-azacytidine and Akt inhibitor VIII from Calbiochem (Darmstadt, Germany). The human monoclonal anti-DR4 agonistic IgG antibody R1-B12 was kindly provided by Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan).
3
Western Blot Analysis of B7-H6 and Downstream Signaling
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Cell lysates were prepared by adding 2x sample buffer, followed by ultrasonic sonication. WB analysis was performed according to standard protocol by separating the cell lysates with SDS-PAGE, followed by wet transfer to a PVDF membrane (Merck Millipore, Billerica, MA, USA). A rabbit anti-human B7-H6 multiclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-human phosphorylated-Akt antibody, anti-pan-Akt antibody, anti-phosphorylated-ERK antibody, anti-pan-ERK monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-human GAPDH monoclonal antibody (Multi Sciences, Hangzhou, China) were incubated separately at corresponding recommended concentration overnight at 4°C. Finally, HRP-conjugated goat anti-rabbit and goat anti-mouse H + L chain polyclonal antibodies (Multi Sciences, Hangzhou, China) were incubated at RT for an hour at a concentration of 1 : 10,000. The blots were visualized with an ECL detection kit (Bio-Rad, Hercules, CA, USA) and imaged by a ChemiScope system (Model No. 6300).
4
Protein Expression and Quantification
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Cells were lysed in RIPA lysis buffer (Millipore, Billerica, MA), containing a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations in the lysates were determined by bicinchoninic acid assay. The lysates were then added with SDS-PAGE sample buffer and 10-18 mg proteins were subjected to 10% SDS-PAGE and western blot analysis using anti-flag antibody (Sigma-Aldrich), anti-insulin receptor antibody, anti-Akt antibody, anti-phosphorylated Akt antibody, anti-Erk antibody, and anti-phosphorylated Erk antibody (Cell Signaling Technology, Beverly, MA). The immune complex was detected using the ECL Advance Western Blot Detection System (GE Healthcare, Buckinghamshire, UK). Signal intensity was quantitated by ImageJ (distributed by NIH).
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