Infected cells were washed two times and lysed in ice-cold RIPA buffer for 0.5 h. An equal amount of cell lysates was separated using SDS-PAGE, followed by transferring them to the PVDF membrane (Millipore, Billerica, MA, USA). The transferred membrane was blocked in 5% non-fat milk (Sangon, Shanghai, China), followed by incubation with the indicated primary antibodies at 4°C overnight. Finally, the membrane was washed three times with TBST and incubated with relevant HRP-conjugated secondary antibodies (Cell Signaling Technology Company, Danvers, MA, USA) at room temperature for one hour and visualized using the BeyoECL Star kit (Beyotime, Shanghai, China). The primary antibodies, viz anti-ERK1/2 (AM076), anti-p38 (AM065), anti-JNK (AJ518), anti-p65 (AN365), and anti-Myd88 (AF2116), were obtained from Beyotime Company (Shanghai, China). The primary antibodies, viz anti-IRAK4 (#4363), anti-phospho-p38 (#4511), anti-phospho-JNK (#4511), anti-phospho-ERK1/2 (#4370), anti-phospho-p65 (#3033), anti-cleaved caspase-3 (#9664), anti-cleaved caspase-9 (#9505), anti-cleaved caspase-8 (#9748), anti-phospho-IRAK4 (#11927), and anti-β-actin (#4970S), were obtained from Cell Signaling Technology Company. ImageJ image software was used to quantify the bands and β-actin as an internal control.
Anti phospho irak4
Manufactured by Cell Signaling Technology
Anti-phospho-IRAK4 is a primary antibody that detects phosphorylated IRAK4, a key component in the Toll-like receptor and IL-1 receptor signaling pathways. This antibody can be used to monitor the activation status of IRAK4.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Beyoecl star kit, by Beyotime (1 mentions)
Non fat milk, by Sangon (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Anti irak4, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti ikkβ, by Cell Signaling Technology (1 mentions)
Anti phospho ikkα β, by Cell Signaling Technology (1 mentions)
Cryo 2, by Adipogen (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Anti total p65, by Cell Signaling Technology (1 mentions)
Ecl prime reagent, by Cytiva (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
2 protocols using anti phospho irak4
1
Western Blot Analysis of Cellular Signaling
2
Western Blot Analysis of NLRP3 and NF-κB Signaling
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Cell lysates were collected in radioimmunoprecipitation buffer (150 mmol/L NaCl, 1% IGEPAL, 0.5% deoxycholic acid, 0.1% SDS, 50 mmol/L Tris, pH 7.5) containing protease inhibitor cocktail (Complete, mini, EDTA-free; Roche). Samples were separated on 8% SDS-PAGE gels then transferred to nitrocellulose membranes (Amersham Biosciences).
Membranes were blocked with 5% skim milk and probed with anti-NLRP3 antibody (Cryo-2; Adipogen), anti-phospho-p65, anti-total p65, anti-phospho-IKKα/β, anti-IKKβ, anti-phospho-IRAK4, anti-total IRAK4 or anti-β-actin (all from Cell Signaling). Bound primary antibodies were detected with HRP-conjugated anti-rabbit (Dako). Labelled proteins were visualized by incubating the membrane in ECL Prime reagent (Amersham Biosciences) and using an ImageQuant LAS 4000 (GE Healthcare). Sodium fluoride was added to radioimmunoprecipitation buffer and skim milk at 20 mM or to other buffers at 2 mM when assessing phosphorylation. Densitometry was performed using ImageJ.
Membranes were blocked with 5% skim milk and probed with anti-NLRP3 antibody (Cryo-2; Adipogen), anti-phospho-p65, anti-total p65, anti-phospho-IKKα/β, anti-IKKβ, anti-phospho-IRAK4, anti-total IRAK4 or anti-β-actin (all from Cell Signaling). Bound primary antibodies were detected with HRP-conjugated anti-rabbit (Dako). Labelled proteins were visualized by incubating the membrane in ECL Prime reagent (Amersham Biosciences) and using an ImageQuant LAS 4000 (GE Healthcare). Sodium fluoride was added to radioimmunoprecipitation buffer and skim milk at 20 mM or to other buffers at 2 mM when assessing phosphorylation. Densitometry was performed using ImageJ.
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