Cells were washed with PBS three times, and total protein was immediately extracted with RIPA lysis buffer (Biossci, Wuhan, China) on the ice. After the loading buffer was added, the protein samples were heated in boiling water for 10 min. Next, 20 µg of samples in each group was separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). After that, the membrane was blocked with skim milk, and incubated with the primary and secondary antibodies, and the protein bands were subsequently visualized with the enhanced chemiluminescence detection kit (Tanon, Shanghai, China). The antibodies included: anti-IRAK1 (Abcam, ab180747, 1:1000), anti-GAPDH (Abcam, ab8245, 1:3000), and secondary antibody (Proteintech, Wuhan, China, 1: 5000).
Ab8245
Manufactured by Proteintech
Sourced in United States
Ab8245 is a primary antibody produced by Proteintech. It is a tool used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab180747, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Blocking buffer, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Bioss Antibodies (1 mentions)
Tanon 2500, by Solarbio (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one image analysis software, by Bio-Rad (1 mentions)
Ab125066, by Proteintech (1 mentions)
Ab128870, by Abcam (1 mentions)
Ab28481, by Abcam (1 mentions)
Ab184032, by Abcam (1 mentions)
Ab45174, by Abcam (1 mentions)
Pvdf membrane, by Pall Corporation (1 mentions)
4 protocols using ab8245
1
Western Blot Analysis of IRAK1 Protein
2
Protein Expression Analysis via Western Blot
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The proteins in this study were lysed using RIPA Lysis Buffer (C05-01001, Bioss, China), followed by quantified with BCA Kit (A53225, ThermoFisher, USA). 20 μg of protein was taken to perform electrophoresis with SDS-PAGE, and then transferred to PVDF membrane (IPVH00010, Millipore, USA). After sealing with blocking Buffer (37565, Thermo Scientific) for 2 h, membranes were then incubated with primary antibodies (4°C, overnight). Afterwards, membranes were incubated with secondary antibodies. Protein bands were visualized with ECL luminous fluid (WBKlS0100, Millipore) and analyzed with gel imaging system (Tanon 2500, Solarbio, China) and Quantity One image analysis software (Bio-Rad, USA). All antibodies produced by Abcam (UK) were as follows: E-Cadherin (ab40772, 1/10000, 97 kDa); N-Cadherin (ab18203, 1/10000, 130 kDa); Vimentin (ab92547, 1/1000, 54 kDa); E2F7 (24489-1-AP, 1/1000, 90 kDa, Proteintech, USA); GAPDH (ab8245, 1/5000, 36 kDa); Goat Anti-Rabbit (ab205718); Goat Anti-Mouse (ab205719).
3
Western Blotting Antibody Validation
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A Western blotting assay was carried out following our previous method [25 (link)]. Information about the antibodies is as follows: anti-PKCiota antibodies (BD Biosciences, Franklin Lakes, NJ, USA, 610175, 1:1000 dilution; Proteintech, Wuhan, China, 13883-1-AP, 1:1000 dilution), anti-GPX4 antibodies (Abcam, Cambridge, UK, ab125066, 1:2000 dilution; Proteintech, 67763-1-Ig, 1:1000 dilution), anti-USP14 antibodies (Cell signaling technology, Boston, USA, 11931S, 1:1000 dilution; Proteintech, 14517-1-AP, 1:2000 dilution), anti-p62 antibody (MBL, M162-3, 1:1000 dilution), anti-K63 linkage-specific polyubiquitin antibody (Cell Signaling technology, 5621, 1:1000 dilution), anti-p-AKT antibody (Cell Signaling technology, 4060, 1:2000 dilution), anti-AKT antibody (Cell Signaling technology, 4691, 1:1000 dilution), anti-CANX antibody (Proteintech, 10427-2-AP, 1:5000 dilution), anti-SOX2 antibody (Proteintech, 11064-1-AP, 1:1000 dilution), and anti-GAPDH antibodies (Abcam, ab8245, 1:2000 dilution; Proteintech, 10494-1-AP, 1:5000 dilution). Measurements were carried out in biological triplicates.
4
Western Blot Analysis of Cellular Proteins
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Equal amounts of proteins were separated on a 10% pre-cast SDS-PAGE (Genscript, Nanjing, China), and then transferred to polyvinylidene difluoride (PVDF) membranes (PALL Life Sciences, USA). The membranes were blocked with 5% milk in TBST (TBS with 0.1% Tween-20) for 1 h at room temperature, and then incubated overnight at 4 ℃ with primary antibodies: anti-Rbbp4 antibody (Proteintech, 20364-1-AP), anti-Tcea1 antibody (Proteintech, 17825-1-AP), anti-ILF2 antibody (Proteintech, 14714-1-AP), anti-NPM1 antibody (Proteintech, 10306-1-AP), anti-Acc1 antibody (abcam, ab45174), anti-Fasn antibody (abcam, ab128870), anti-Srebp1c antibody (abcam, ab28481), anti-Acox1 antibody (abcam, ab184032), anti-Cpt1α antibody (abcam, ab128568), anti-Flag antibody (Proteintech, 20543-1-AP), anti-GAPDH antibody (abcam, ab8245), anti-β-actin antibody (Proteintech, 66009-1-Ig). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Protein bands were visualized using enhanced chemiluminescence kit (UUbio, Suzhou, China) with a Tanon 5200 multi-imaging system (Tanon Technology Co., Ltd, Shanghai, China).
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