Proteins were separated on gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (7.5–15% acrylamide) and electrophoretically transferred to polyvinylidene difluoride membrane. Membranes were blocked using 5% skim milk in PBS plus 0.05% v/v Tween-20 before incubation with anti-phospho p44/42 MAP kinase antibody (9101; Cell Signaling) and anti-GAPDH antibody (sc-47724; Santa Cruz Biotech, Dallas, TX). HRP-conjugated secondary antibodies and ECL plus kit (Amersham Life Sciences, Piscataway, NJ) were used to detect proteins of interest.
Anti phospho p44 42 map kinase antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phospho-p44/42 MAP kinase antibody is a laboratory tool used to detect the phosphorylated form of the p44/42 mitogen-activated protein (MAP) kinase. This antibody recognizes the active, phosphorylated state of these kinases, which are important signaling molecules involved in cellular processes such as proliferation, differentiation, and survival.
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Lab products found in correlation
Protease inhibitor cocktail, by Sangon (2 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Blocking reagent, by Roche (1 mentions)
Rabbit anti gfp alexa 488, by Thermo Fisher Scientific (1 mentions)
Rat anti α tubulin antibody, by Bio-Rad (1 mentions)
Mpk1 antibody n terminal anti mpk1, by Santa Cruz Biotechnology (1 mentions)
Phospho ser235 ser236 s6, by Cell Signaling Technology (1 mentions)
Anti mouse antibody, by LI COR (1 mentions)
Anti p44 42 map kinase antibody, by Cell Signaling Technology (1 mentions)
Anti gfp antibody, by Abmart (1 mentions)
Blotting apparatus, by Bio-Rad (1 mentions)
7 protocols using anti phospho p44 42 map kinase antibody
1
Western Blot Analysis of Phospho-p44/42 MAPK
2
Phospho-p44/42 MAPK Localization in Stab2 Morphant Embryos
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Control uninjected and Stab2 morphant embryos were fixed in 4% paraformaldehyde (PFA) at the 20 somite stage, dehydrated in ethanol and stored at −20°C. Embryos were rehydrated and blocked in blocking reagent (Roche). Following blocking, embryos were incubated in rabbit polyclonal anti-phospho-p44/42 MAP kinase antibody (Cell Signaling Technologies) overnight, washed, incubated in rabbit anti-Alexa647 (Invitrogen) for four hours, washed, and incubated in rabbit anti-GFP Alexa-488 (Invitrogen) overnight. Embryos were analyzed and imaged using fluorescent microscopy.
3
Western Blot Analysis of MAPK Phosphorylation
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To detect phosphorylated Mkc1 and Cek1 as well as α-tubulin, cells of an overnight culture were adjusted to an OD600 of 0.5 in SD medium (control) or SD medium supplemented with 450 µg/ml congo red, and incubated for 4 hours at 30°C. Cell disruption, protein extraction and western blot analysis using anti-phospho-p44/42 MAP kinase antibody (Cell Signalling Technology) and rat anti-α-tubulin antibody (AbD Serotec), respectively, were performed as previously described [87] (link).
4
Rapamycin-Induced Mps1 and TOR Phosphorylation
5
Detecting Activated MAPKs and ROS in Arabidopsis
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For the detection of activated MAPKs, immunoblot analyses using the anti-phospho p44/42 MAP kinase antibody (Cell Signaling Technology) were performed as described (Zhang et al., 2013 (link)). The detection of ROS in Arabidopsis leaf pieces was performed as described (Wan et al., 2019 (link)). Callose deposition in Arabidopsis leaves was stained with aniline blue 24 h after MAMP infiltration as described (Gomez-Gomez et al., 1999 (link)). Digital pictures of stained leaves were taken under UV-light with the microscope and callose depositions were quantified by counting light, fluorescent pixels with the Adobe Photoshop CS “select range” tool. Per treatment at least 10 pictures of at least 5 different leaves were analyzed.
For transcript profiling, RNA isolation and RT-qPCR analysis of plant material were performed as described previously (Zhang et al., 2013 (link)). The sequences of the primers used for PCR amplifications are listed inSupplementary Table S1 .
For transcript profiling, RNA isolation and RT-qPCR analysis of plant material were performed as described previously (Zhang et al., 2013 (link)). The sequences of the primers used for PCR amplifications are listed in
6
Protein Phosphorylation Analysis in Fungal Mutants
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The mutants and Guy11 strains were cultured in liquid CM for 2 days and then the total proteins were isolated from vegetative hyphae as described by Bruno et al. (2004 (link)). For protein phosphorylation analysis, the proteinase inhibitor cocktail (cOmplete; Sigma‐Aldrich) was added. The intensity of the signal corresponding to phosphorylated MoMps1 was detected by binding of an antiphospho‐p44/42MAP kinase antibody (Cell Signaling Technology), and the anti‐p44/42 MAP kinase antibody (Cell Signaling Technology) was used as control. For green fluorescent protein (GFP)‐tagged protein detection, samples were analysed by 8% SDS‐PAGE followed by western blotting with the anti‐GFP antibody (Abmart) and the anti‐mouse antibody (LI‐COR, IRDye), followed by detection using the ODYSSEY infrared imaging system (application software v. 2.1).
7
Quantifying Phosphorylated Mps1 Levels
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The ΔMomaf1 mutant and wild-type strains were cultured in liquid CM for 2 days and then harvested, and 1 mL of protein lysis buffer and 10 µL of protease inhibitor cocktail (Sangon, Shanghai, China) were added. After vortexing and homogenization, the lysate was centrifuged at 12,000 rpm for 10 min at 4 °C. Then, 200 μL of the supernatant was mixed with 50 μL loading buffer and boiled for 5 min. Obtained proteins were separated on SDS–PAGE gels and transferred onto a polyvinylidene fluoride membrane using a Bio-Rad blotting apparatus. The intensity of the phosphorylated Mps1 signal was detected by the addition of an anti-phospho-p44/42 MAP kinase antibody (Cell Signaling Technology, Boston, MA, USA), with an anti-MAPK1 antibody (N-terminal anti-Mpk1) used as a control.
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