Rabbit antibodies against human p-TBK1 Ser172 (catalog no. 5483), TBK1 (catalog no. 3504), IRF3 (catalog no. 11904), p-STING Ser366 (catalog no. 85735), and STING (catalog no. 13647) were obtained from Cell Signaling Technology. Rabbit antibodies against human p-IRF3 Ser386 (ab76493), MAVS (ab31334), and green fluorescent protein (GFP) (ab290) were purchased from Abcam. Rabbit antibody against HA was purchased from HUAXINGBIO (HX1820). Mouse antibodies against laminB1 (sc-365962) were purchased from Santa Cruz Biotechnology. Mouse anti-FLAG antibody (M2) (F1804), M2-conjugated agarose (A2220), mouse anti-HA antibody (H3663), and anti-HA–conjugated agarose (A2095) were purchased from Sigma-Aldrich. Rabbit anti-PPM1G antibody (A300-881A) was purchased from Bethyl Laboratories. Poly(I:C) and poly(dA:dT) were purchased from InvivoGen (tlrl-pic, tlrl-patn-1). Mouse anti-ORF33 antibody was provided by F. Zhu (Florida State University, USA). Mouse anti-K8/KbZIP and anti-ORF45 antibodies were provided by Y. Yuan (University of Pennsylvania, USA). Human IFNβ ELISA Kit was purchased from R&D Systems (DIFNB0). Duolink In Situ–Fluorescence kit for PLA was purchased from Sigma-Aldrich (DUO92008, DUO92002, and DUO92004).
Ab31334
Manufactured by Abcam
Sourced in United States
Ab31334 is a laboratory equipment product offered by Abcam. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Roche (2 mentions)
Ab76493, by Abcam (1 mentions)
Human ifn β elisa kit, by R&D Systems (1 mentions)
P tbk1 ser172, by Cell Signaling Technology (1 mentions)
P sting ser366, by Cell Signaling Technology (1 mentions)
Sc 365962, by Santa Cruz Biotechnology (1 mentions)
Tlrl patn 1, by InvivoGen (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Tlrl pic, by InvivoGen (1 mentions)
Poly da dt, by InvivoGen (1 mentions)
Ab69983, by Abcam (1 mentions)
Lsrii analyzer, by BD (1 mentions)
Alexa fluor goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Sd 001, by Invent Biotechnologies (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Sc 376845, by Santa Cruz Biotechnology (1 mentions)
Protein a g agarose, by Beyotime (1 mentions)
Ab46154, by Cell Signaling Technology (1 mentions)
Wbkls0500, by Bio-Techne (1 mentions)
Flour chem fc2 imaging system, by Bio-Techne (1 mentions)
6 protocols using ab31334
1
Deciphering Innate Immune Signaling
2
Viral Sensor and SETDB1 Regulation
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Cells were transduced with viral sensor sgRNAs (sgRNAs are in Table 2 ). 2 d after transduction, cells were subjected to a second transduction with SETDB1-specific sgRNAs or an NTC control sgRNA. Cells were selected with 1 µg/ml puromycin (viral sensor sgRNAs) and 5 µg/ml blasticidin (SETDB1 or NTC sgRNAs). Cells were harvested for FACS analysis 14 d after the second transduction. In brief, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed with 1 ml PBS. Cells were permeabilized with PBS + Tween 0.1% for 15 min. Primary antibodies were used at a 1:100 dilution (MAVS; ab31334; Abcam; MDA5; ab69983; Abcam) in 150 µl FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) at 4°C for 30 min. The cells were washed twice with 1 ml FACS buffer. The secondary was Alexa Fluor goat anti–rabbit IgG (H + L; A11008; ThermoFisher) and was used at a 1:500 dilution in 150 µl FACS buffer. Cells were washed twice with 1 ml FACS buffer and analyzed on a LSRII analyzer (BD).
3
Protein Expression Analysis in Blood and Lymph Nodes
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The protein was extracted from peripheral serum using a serum protein extraction kit (BB-3138-2, BestBio), and the protein was extracted from lymph nodes using a the total protein extraction kit (SD-001, invent biotechnologies). The expression of CCR5 (ab1673, Abcam) and MAVS (ab31334, Abcam) in peripheral blood and LNs was detected by Western blot. This was achieved with a rabbit α-syn polyclonal antibody diluted at 1:1000 and then with the appropriate peroxidase-conjugated secondary antibodies diluted at 1:2000. Normalization was made against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression diluted at 1:1000 (Santa Cruz biotechnology, Dallas, TX). The developed films were scanned as tiff images in 8-bit gray-scale format at a setting of 300 dpi and the band intensities were measured by image J software (National Institutes of Health, Bethesda, MD)
4
Immunoprecipitation of MAVS and RIG-I
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Mouse lung tissues and A549 cells were lysed in RIPA buffer as mentioned for Western blotting. The lysates were centrifuged at 12,000×g and 4 °C for 15 min. Supernatants were collected, and 400 μg lysate samples were incubated with 2 μg of an anti-MAVS primary antibody overnight at 4 °C in a rotator. Then, 20 μL Protein A/G agarose (Beyotime Biotechnology, Shanghai, China) was added and incubated for an additional 3 h at 4 °C, after which the lysates were washed 5 times with PBS and subjected to Western blot analysis with primary antibodies specific for MAVS (Abcam, Source: rabbit, Catalog: ab31334, Abcam, Cambridge, MA, USA) and RIG-I (Santa Cruz, Source: mouse, Catalog: sc-376845, Santa Cruz, CA, USA).
5
Western Blot Analysis of Immune Signaling Proteins
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Cells were homogenized in cell lysis buffer (Beyotime), and protein concentration was determined using a BCA kit (Beyotime). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with specific primary antibodies. Then the membrane was incubated with appropriate HRP-conjugated secondary antibodies. Signals were monitored by an enhanced chemiluminescence reagent (Millipore, WBKLS0500) and subjected to Alpha Innotech Flour Chem-FC2 imaging system (Alpha Innotech). The antibodies used in this study are as follows: VEGF (abcam, ab46154), RIG-I (Cell signaling technology, 3743), MAVS (abcam, ab31334), NOXA (abcam, ab13654), GAPDH (Bioworld Technology, MB001), β-actin (Cell signaling technology, 4967).
6
Western Blot Analysis of Cellular Proteins
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Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany, 11873580001). Protein concentration was determined. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with specific primary antibodies, followed by incubation with appropriate horseradish peroxidase–conjugated secondary antibodies. Signals were developed using an enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany, WBKLS0500) and captured on an Alpha Innotech Fluor Chem FC2 imaging system (Alpha Innotech, San Leanardo, CA). Antibodies used in this study were: rabbit anti-β-ACTIN (Biosynthesis Biotechnology, Beijing, China, bs0061R, 1:1000), rabbit anti-HMGB1 (Abcam, Hong Kong, China, ab191583, 1:1000), rabbit anti-AMPK/pAMPK (Cell Signaling Technology, Danvers, MA, #2532 / #2531, 1:1000), rabbit anti MAVS (Abcam, ab31334, 1:500) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000).
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