20 MTs pairs per genotype from female flies were dissected in PBS, placed in 30 μl 2xSDS sample buffer (Thermo Scientific, #39001) containing 5% 2-Mercaptoethanol at 100 °C for 10 minutes, ran 4ul on a 4%-20% polyacrylamide gel (Bio-Rad, #4561096), and transferred to an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). Membranes were blocked by 5% skim milk in 1x Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) at room temperature for 30 minutes. The following primary antibodies were used: mouse anti-tubulin (Sigma, T5168, 1:10,000), rabbit anti-JNK Antibody (D-2) (Santa Cruz, sc-7345, 1:1000), rabbit phospho-JNK (Cell Signaling, 4668 T, 1:1000), rabbit anti-ERK Antibody (Cell Signaling, 4695, 1:1000), rabbit phospho-ERK (Cell Signaling, 4370, 1:1000). After washing with TBST, signals were detected with enhanced chemiluminescence (ECL) reagents (Amersham, RPN2209; Pierce, #34095). Western blot images were acquired by Bio-Rad ChemiDoc MP.
Rabbit anti erk antibody
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
The Rabbit anti-ERK antibody is a primary antibody that specifically recognizes extracellular signal-regulated kinase (ERK), a family of serine/threonine protein kinases that play a crucial role in various cellular processes. This antibody is produced in rabbits and can be used for the detection and analysis of ERK in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti perk antibody, by Cell Signaling Technology (3 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
2xsds sample buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon p polyvinylidene fluoride, by Merck Group (1 mentions)
Rabbit phospho erk, by Cell Signaling Technology (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Rabbit anti klf4 antibody, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Rabbit anti gapdh antibody, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Mouse anti β actin antibody, by Abcam (1 mentions)
Rabbit anti phospho erk, by Cell Signaling Technology (1 mentions)
Rabbit anti jnk antibody, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit anti erk antibody
1
Protein Expression Analysis in Drosophila
2
Quantitative Western Blot Analysis of ESC Transcription Factors
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ESCs were lysed and western blotting was performed as described previously [22 (link)]. Membranes were immunoblotted with rat anti-mouse Klf5 antibody (KM1784; Kyowa Kirin), rabbit anti-pERK antibody (Cell Signaling Technology), rabbit anti-ERK antibody (Cell Signaling Technology), rabbit anti-Klf2 antibody (Millipore), rabbit anti-Klf4 antibody (Abcam), or anti-β-actin antibody (MBL), followed by an appropriate secondary antibody [horseradish peroxidase-conjugated rabbit anti-mouse IgG (Invitrogen) or horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymax)]. Immunoreactive proteins were detected using enhanced chemiluminescence (Chemilumi One; Nakalai) and an ImageQuant LAS 4000 imager (GE Healthcare). Signal intensity of western blotting was quantified using ImageJ. Signals were normalized to the intensity of ERK or β-actin.
3
Quantifying Phosphorylated ERK Levels
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Total protein was collected by washing cells twice with ice cold PBS and subsequent lysis in RIPA buffer (20 mM Tris-HCl (pH 7.5), 1% Triton X-100 (Sigma), 137 mM NaCl, 10% glycerol and protease inhibitors (Roche)). Concentration of the protein was measured with the Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. SDS-Page was used to separate the proteins, after which they were transferred onto a nitrocellulose membrane. Membranes were probed with a rabbit anti-pERK antibody (Cell Signaling, product nr #4376S), a rabbit anti-ERK antibody (Cell Signalling, product nr#9102) and a rabbit anti-GAPDH antibody (Abcam, Cambridge, UK, product nr ab9385), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz, product nr sc-2030)Bound antibodies were detected using chemiluminescence (ECL, GE Healthcare, Chalfont St. Giles, UK). Afterwards, band intensity was measured using ImageJ software (imagej.nih.gov/ij/ ) and pERK band intensity was corrected for corresponding GAPDH band intensity.
4
Ano1 and TRPV3 Inhibitor Assay Protocol
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T16Ainh-A01 (T16A, Calbiochem) and Ani9 (Sigma-Aldrich) were used as an Ano1 inhibitor. Dyclonine (MedChemExpress) was used as a TRPV3 inhibitor. The following antibodies were used: rabbit anti-ANO1 antibody (Abcam, ab53213, 1:5 for Western blotting), (Abcam, ab53212, 1:100 for immunoprecipitation), rabbit anti-phospho-ERK (extracellular signal-related kinase) antibody (Cell Signaling Technology, #4370, 1:1000), rabbit anti-phospho-p38 antibody (Cell Signaling Technology, #4511, 1:1000), rabbit anti-phospho-JNK (c-Jun N-terminal Kinase) antibody (Cell Signaling Technology, #4668, 1:1000), rabbit anti-ERK antibody (Cell Signaling Technology, #4695, 1:1000), rabbit anti-p38 antibody (Cell Signaling Technology, #8690, 1:1000), rabbit anti-JNK antibody (Cell Signaling Technology, #9252, 1:1000), mouse anti-β-actin antibody (Abcam, ab6276, 1:2500), rabbit anti-TRPV3 antibody (Cell Signaling Technology, #3484, 1:1000 for Western blotting; 1:50 for immunoprecipitation), anti-rabbit-HRP antibody (Cell Signaling Technology, #7074, 1:2000) and anti-mouse-HRP antibody (Cell Signaling Technology, #7076, 1:2000).
5
Western Blot Analysis of Signaling Proteins
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The details of the experimental procedures were described previously.35 (link) The antibodies used in this assay were the following: mouse anti-ZNF32 antibody (1:200), mouse anti-TGF-βR2 antibody (1:100), rabbit anti-β-actin antibody (1:800, Santa Cruz Biotechnology), rabbit anti-ERK antibody (1:1000, Cell Signaling Technology, Lexington, KY, USA), rabbit anti-p-ERK antibody (1:500, Cell Signaling Technology), rabbit anti-SMAD2 antibody (1:500, Cell Signaling Technology), rabbit anti-pSMAD2 antibody (1:200, Cell Signaling Technology), rabbit anti-MEK antibody (1:1000, Cell Signaling Technology), rabbit anti-p-MEK antibody (1:500, Cell Signaling Technology), horseradish peroxidase-conjugated secondary antibody to rabbit IgG (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish peroxidase-conjugated secondary antibody to mouse IgG (1:8000, Santa Cruz Biotechnology).
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