The procedure for immunoblotting analyses was performed as previously described.21 (link) The following antibodies were used for immunoblotting analyses: cGAS (Novus, NBP1-86761), IRF3 (HUABIO, ET1612-14), P65 (CST, 6956), p-IRF3 (Abcam, ab76493), p-P65 (CST, 3033), HIF-1α (CST, 14179), β-actin (Santa Cruz, sc-47778), anti-mouse IgG (CST, 7076) and anti-rabbit IgG (CST, 7074). Protein bands were visualized by a Tanon 5200 chemiluminescence image analysis system (China).
P irf3
Manufactured by Abcam
P-IRF3 is a protein that functions as a transcription factor. It is involved in the regulation of genes associated with the immune response and inflammation. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (1 mentions)
Sting, by Abcam (1 mentions)
Gsdmd, by Santa Cruz Biotechnology (1 mentions)
Casp9, by Abcam (1 mentions)
Acsl4, by Abcam (1 mentions)
P tbk1, by Abcam (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Ab3114, by Santa Cruz Biotechnology (1 mentions)
Odyssey software, by LI COR (1 mentions)
Amersham protran membrane, by GE Healthcare (1 mentions)
H6908, by Abcam (1 mentions)
Secondary abs, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
F7425, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
P tbk1, by Cell Signaling Technology (1 mentions)
Immobilon western chemilum hrp substrate, by Merck Group (1 mentions)
4 protocols using p irf3
1
Immunoblotting Analysis of Immune Signaling
2
Western Blot Analysis of Cell Signaling
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Tissue lysates were homogenized in SDS lysis buffer [Cell Signaling Technology (CST), #7722] and transferred onto polyvinylidene difluoride membranes. After blocking in 5% milk in tris-buffered saline and 0.1% Tween 20, membranes were incubated overnight with the following antibodies at 4°C: CASP9 (Abcam, #ab202068), cleaved CASP3 (CST, #9664), RIP3 (Millipore Sigma, #PRS2283), BAX (CST, #2772), BCL2 (CST, #3498), GSDMD (Santa Cruz Biotechnology, sc-393656), NLRP3 (CST, #15101), ACSL4 (Abcam, #ab155282), LC-3 (CST, #2775S), cGAS (CST, #31659), STING (CST, #13647S), TBK1 (CST, #38066), pTBK1 (CST, #5483), IRF3 (CST, #4302), pIRF3 (CST, #4947), p65 (CST, #8242), pp65 (CST, #3033), and GAPDH (CST, #5174). Membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibody (CST, #7074 and #7076) for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, #34094; Thermo Fisher Scientific). Western blots were quantified using the Fiji software (58 (link)).
3
Western Blot Analysis of Cellular Proteins
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Proteins in cell lysates were separated by electrophoresis and transferred to a nitrocellulose Amersham Protran membrane (GE Healthcare). The membrane was blocked for 1 hr in 5% milk in PBS containing 0.1% Tween-20 at 23°C. Then, the membrane was incubated with primary antibody (Ab) diluted in blocking buffer at 4°C overnight. Antibodies used were from the following sources: DNA-PKcs (NeoMarkers, MS-423-P1, diluted 1:1000), Ku70 (AbCam, ab3114, diluted 1:1000), Ku80 (Santa Cruz, sc-1484, 1:500), FLAG (Sigma-Aldrich, F7425, diluted 1:5000), HA (Sigma-Aldrich, H6908, diluted 1:1000), P-IRF3 (Abcam, ab76493, diluted 1:1500), and α-tubulin (Merck Millipore, 05-829, diluted 1:10000). Membranes were probed and visualized with LI-COR Biosciences secondary Abs and the Li-Cor Odyssey infrared imaging system according to the manufacturer’s instructions. A quantification of protein bands was performed, where indicated, by using Odyssey software (LI-COR Biosciences).
4
Western Blot Analysis of Immune Signaling
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Whole-cell extracts were prepared by lysis and sonication of cells in RIPA buffer (50 mM TRIS, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% Sodium deoxycholate, 2 mM EDTA) and then analyzed using standard SDS-PAGE procedures. The primary antibodies were incubated in TBST buffer with 5% BSA over night at 4 °C. The second antibodies were incubated in TBST buffer for 1 h at room temperature. Proteins were visualized with the immobilon western chemilum HRP substrate (Millipore) and imaged by using the Chemiluminescence Imaging System. Antibodies p65 (Cat# 4764, RRID: AB_823578), pP65 (Cat# 3033), TBK1 (Cat# 3504, RRID: AB_2255663), P-TBK1 (Cat# 5483 S), cGAS (Cat# 15102) were purchased from Cell signaling technology. IRF3 (Cat# Ab25950), p-IRF3 (Cat# Ab138449) were purchased from Abcam. Actin was purchased from Proteintech.
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