Total cell protein lysates were extracted from the transfected or infected cells using the ProteinExt mammalian total protein extraction kit (TransGen). The total protein concentration was determined with a bicinchoninic acid protein assay kit (CWBIO, Beijing, China). The cellular proteins were separated with 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Freiburg, Germany). Each PVDF membrane was blocked with 5% (wt/vol) skim milk and 0.1% Tween 20 in Tris-buffered saline (TBST) for 2 h at room temperature and then incubated overnight at 4°C with a primary antibody. The following antibodies were used in the experiments: anti-IBV-N (mouse; 3BN1; HyTest, Turku, Finland), anti-PKR (rabbit; Ab32506; Abcam, Cambridge, UK), anti-phospho-T446 PKR (rabbit; Ab32036; Abcam), and anti-β-actin (mouse; 3700S; Cell Signaling Technology, Danvers, MA, USA). The membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody and diluted at 1:10,000 for 1 h at room temperature. HRP was detected with Western lightning chemiluminescence reagent (CWBIO).
Ab32506
Manufactured by Abcam
Sourced in United Kingdom
Ab32506 is a laboratory equipment product. It is designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid protein assay kit, by CWBIO (1 mentions)
Ab32036, by Abcam (1 mentions)
Proteinext mammalian total protein extraction kit, by Transgene (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Ab75749, by Abcam (1 mentions)
Anti phospho p65 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho jnk antibody, by Cell Signaling Technology (1 mentions)
Westernbright quantum kit, by Advansta (1 mentions)
Anti phospho p38 antibody, by Cell Signaling Technology (1 mentions)
Anti jnk antibody, by Cell Signaling Technology (1 mentions)
Pvdf blocking agent, by Toyobo (1 mentions)
Anti total p 38 antibody, by Cell Signaling Technology (1 mentions)
Ab126630, by Abcam (1 mentions)
Ab81303, by Abcam (1 mentions)
Rnase 1, by Promega (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa 594, by Santa Cruz Biotechnology (1 mentions)
Rnase 3, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit alexafluor 594, by Thermo Fisher Scientific (1 mentions)
4 protocols using ab32506
1
Western Blot Analysis of Viral and Host Proteins
2
Western Blot Analysis of Signaling Proteins
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Protein samples were reduced with DTT and loaded onto NuPAGE Bis-Tris gels to detect total and phosphorylated JNK, p-38, and p-65, HSP90A, PKR, and PACT and onto NuPAGE Tris-Acetate gels to detect AHNAK. For immunodetection, gels were transferred onto PVDF membranes. The membranes were blocked with PVDF blocking agent (Toyobo, Osaka, Japan), and then incubated with anti-JNK antibody (#9252; Cell Signaling Technology, Danvers, MA), anti-phospho-JNK antibody (#4668; Cell Signaling Technology), anti-total p-38 antibody (#8690; Cell Signaling Technology), anti-phospho-p-38 antibody (#4511; Cell Signaling Technology), anti-total p65 antibody (#8242; Cell Signaling Technology), anti-phospho-p65 antibody (#3033, Cell Signaling Technology), anti-HSP90α antibody (GTX109753; GeneTex, Irvine, CA), anti-AHNAK antibody (ab178317; Abcam, Cambridge, MA), anti-PKR antibody (ab32506; Abcam), or anti-PACT antibody (ab75749; Abcam). The immunoreactive proteins were identified using a Westernbright Quantum Kit (K-12042-D20; Advansta, Menlo Park, CA).
3
Antibody Detection of dsRNA and RLRs
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The antibody used to detect dsRNA was a murine MAb pan-Enterovirus clone 9D5 purchased from Millipore Sigma (cat#3361, ready for use, further diluted by dilution 1:2 in our lab). The rabbit MAbs against PKR (1:200, ab32506), p-PKR (1:100, ab81303) and MDA-5 (1:250, ab126630) were purchased from Abcam. The secondary antibodies, Goat-anti-mouse Alexa Fluor 488 (1:2,000) and Donkey-anti-rabbit Alexa Fluor 594 (1:2,000) were purchased from Invitrogen. The RIG-I antibody conjugated Alexa-594 was purchased from Santa Cruz (1:1,000, sc-376845). A murine monoclonal anti-JUNV NP antibody was obtained from BEI (NA05-AG12) and conjugated to Alexa 647 dyes (1:1,000, Invitrogen). RNase III was purchased from Life technologies (AM2290) and RNase I from Promega (M4261).
4
Western Blot Analysis of RNAi Pathway
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Proteins were extracted from cells and homogenized in 350 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% SDS and Protease Inhibitor Cocktail (complete Mini; Sigma Aldrich). Proteins were quantified by the Bradford method and 20 to 30 μg of total protein extract were loaded on 4–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad). After transfer onto nitrocellulose membrane, equal loading was verified by Ponceau staining. For PVDF membrane, equal loading was verified by Coomassie staining after transfer and blotting. Membranes were blocked in 5% milk and probed with the following antibodies: anti-hDicer (1:500, F10 Santa Cruz, sc-136979) and anti-hDicer (1:1000, A301-937A, Bethyl), anti-TRBP (1:500, D-5 Santa Cruz, sc-514124), anti-PKR (1:2500, Abcam ab32506), anti-PACT (1:500, Abcam, ab75749), anti-HA (1:10000, Sigma, H9658), anti-DHX9 (1:500, Abcam, ab26271), anti-p-eIF2 (1:1000, Ser-52 Santa Cruz, sc-601670), anti-hADAR-1 (1:500 Santa Cruz, sc-271854) anti-p-PKR (1:1000 Abcam ab81303) anti-GFP (1:10000, Roche, 11814460001) and anti-Tubulin (1:10000, Sigma, T6557). Detection was performed using Chemiluminescent Substrate (Pierce, Thermo Fisher Scientific) and visualized on a Fusion FX imaging system (Vilber).
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