Total protein, for the detection of Hog1 or Ypd1, was extracted from the mycelia of the WT or YPD1 KD transformant M. oryzae grown in liquid CM for 72 h, followed by washing and transfer to fresh liquid CM supplemented with or without a stress element for 6 h. The protein samples were electrophoresed on a 15% SDS-polyacrylamide gel, followed by electro-transfer to a PVDF membrane (Hybond-P; GE Healthcare lifesciences). Hybridization with the Ypd1 antibody was studied using a horseradish peroxidase-conjugated secondary antibody followed by detection with SuperSignal West Pico Chemiluminescence substrate (Thermo Scientific). Phosphorylation of Hog1 was studied using anti-phospho p38 MAPK antibody from Peirce Antibodies (Thermo Scientific). An anti-Hog1 antibody (Santa Cruz Biotechnology) was used to detect Hog1 expression as an internal control.
Anti hog1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-Hog1 antibody is a research tool used to detect the Hog1 protein in various cell and tissue samples. Hog1 is a mitogen-activated protein kinase (MAPK) that plays a crucial role in the cellular response to osmotic stress. The Anti-Hog1 antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and study the expression and activation of Hog1 in biological systems.
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Lab products found in correlation
Anti cox 2 antibody, by Thermo Fisher Scientific (2 mentions)
Anti oxa1 antibody, by Santa Cruz Biotechnology (2 mentions)
Anti phospho p38 antibody, by New England Biolabs (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hybond p, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Trans blot membrane, by Bio-Rad (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Zirconia silica bead, by Biospec (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Trans blot nitrocellulose membrane, by Bio-Rad (1 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Anti gfp antibody, by Beyotime (1 mentions)
7 protocols using anti hog1 antibody
1
Western Blot Analysis of Hog1 and Ypd1
2
Western Blot Immunodetection of Mitochondrial Proteins
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From SDS-tricine-PAGE, proteins were electrotransferred onto a nitrocellulose Trans-Blot membrane (Bio-Rad) for immunodetection. Membranes were washed, blocked, and independently incubated for 4 h with the following antibodies: anti-Cox2 antibody at a 1:9000 dilution (Invitrogen; Molecular Probes), anti-Oxa1 antibody at a 1:1000 dilution, anti-Hog1 antibody at a 1:2000 dilution (Santa Cruz Biotechnology), and anti-Atp2 antibody at a 1:50,000 dilution. Alkaline phosphatase-conjugated IgGs (1:15,000 for 2 h) were used as secondary antibodies. Insoluble black–purple precipitates were formed upon addition of nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt. Images of the immunodecorated polypeptide bands were captured in an HP Scanjet G4050. For immunodetection on previously probed membranes using a different primary antibody, membranes were stripped by incubation for 45 min at 50°C in the presence of 2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 100 mM β-mercaptoethanol.
3
Hog1 MAPK Phosphorylation Assay
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Each H99 strain was grown to mid-logarithmic phase in YPD at 30°C. Cultures were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 1% sodium deoxycholate, 5 mM sodium pyrophosphate, 10 nM sodium orthovanadate, 50 mM NaF, 0.1% [w/v] SDS, and 1% [v/v] Triton X-100) containing 1× protease inhibitor cocktail (Calbiochem) with 0.5 mm zirconia/silica beads (BioSpec Products, Inc.) and disrupted. Protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Scientific), and equal amounts of protein were loaded into a 10% Tris-glycine gel (Novex) and transferred to Immuno-blot PVDF membrane (Bio-Rad). A rabbit p38-MAPK-specific antibody (Cell Signaling Technology) was used to detect of phosphorylated Hog1. A rabbit polyclonal anti-Hog1 antibody (Santa Cruz Biotechnology) was used as a loading control.
4
Western Blot Analysis of Mitochondrial Proteins
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From SDS-tricine-PAGE, proteins were electrotransferred onto a nitrocellulose Trans-Blot membrane (Bio-Rad) for immune detection. Membranes were washed, blocked, and independently incubated for 8 h with the following antibodies: anti-Cox1 antibody at a 1:3300 dilution (MitoSciences), anti-Cox2 antibody at a 1:7500 dilution (Invitrogen; Molecular Probes), anti-Cox3 antibody at a 1:15,000 dilution (Molecular Probes), anti-Oxa1 antibody at a 1:1000 dilution, anti-Hog1 antibody at a 1:2000 dilution (Santa Cruz Biotechnology), anti-HA antibody at a 1:15,000 dilution (Sigma Aldrich), and anti-Atp2 antibody at a 1:50,000 dilution. Alkaline phosphatase (ALP)–conjugated immunoglobulin Gs (1:15,000 for 2 h) were used as secondary antibodies. Insoluble black–purple precipitates on bands were formed upon addition of nitro blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt. Images of the immune-decorated polypeptide bands were captured in a HP Scanjet G4050. To carry out a second Western blot reaction with a different primary antibody, membranes were stripped by incubation for 45 min at 50°C in the presence of 2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 100 mM β-mercaptoethanol. Band intensity was measured by densitometric analysis using the GelAnalyzer 2010a freeware.
5
Yeast stress response protein analysis
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Yeast strains were grown in YPD medium to the mid-exponential phase (OD660 = 0.6) and exposed to stress for 10 and 20 minutes. 20 mL of culture was harvested at each step and immediately frozen by immersion in liquid nitrogen. Protein extraction was performed in 0.2 M Tris-HCl buffer pH 7.5 with IGEPAL CA-630 (0.5%), imidazole (10 mM), protease inhibitors (leupeptin, pepstatin A, aprotinin, PMSF) and phosphatase inhibitors (sodium vanadate, sodium fluoride, β-mercaptoethanol) and cells were broken with Mini-BeadBeater-24 as described previously28 (link). Protein extracts (40 µg) were resolved by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred to nitrocellulose blotting membrane (GE Healthcare). PageRuler Prestained Protein Ladder (10 to 180 kDa) was used as a marker. The detection of phosphorylated Hog1 using an anti-phospho-p38 antibody (New England Biolabs) and total levels of Hog1 using an anti-Hog1 antibody (Santa Cruz Biotechnology) was performed as described previously16 (link).
6
Western Blot Analysis of Fusion Proteins
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To confirm the expression of the fusion proteins BcHptK161Q-GFP, BcHptK161R-GFP and BcHptK161K-GFP, Western blotting was carried out using an anti-GFP antibody (Beyotime, Shanghai, China) and anti-acetyl lysine mouse mAb (clone Kac-01, PTM-101) (PTM Biolabs, Hangzhou, China). Conidia were harvested from 10-day old cultures. The mycelia of the mutants were grown in yeast extract peptone dextrose (YEPD) at 25°C for 2 days in a shaker. After the cultures were treated with 20 mM H2O2, 0.2 mg/ml congo red, 1 M NaCl and 1 μg/ml iprodione for 2 h, mycelia were harvested. Protein extraction was carried out as previously described (Gu et al., 2015 (link)). The expression of BcSak1 and phosphorylated BcSak1 was examined by using an anti-Hog1 antibody (Santa Cruz Biotechnology, CA, United States) and an antibody against dually phosphorylated p38 (Thr180/Tyr182) (Cell Signaling Technology, MA, United States).
7
Hog1 Phosphorylation Detection by Western Blot
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Protein extracts were prepared from mid-exponential phase cells and phosphorylated Hog1 was detected by western blot analysis with an anti-phospho-p38 antibody (New England Biolabs) as described previously [6 (link)]. Blots were stripped and total levels of Hog1 were determined by probing with an anti-Hog1 antibody (Santa Cruz Biotechnology), and in some cases protein loading determined using an anti-tubulin antibody (DSHB, University of Iowa).
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