The anti-EMCV 2B antibody (1:1000) was generated by immunizing a rabbit with the C-terminal region of 2B protein, which was supplied by peptide synthesis (FITPPPRFPTISL). Monoclonal antibody against influenza A virus M2 protein (14C2, Cat#ab5416; 1:1000) and anti-dsDNA (35I9 DNA, Cat#ab27156; 1:600) were purchased from abcam. Monoclonal antibody against Flag (M2, Cat#F1804; 1:1000), rabbit polyclonal antibodies against Flag (Cat#F7425; 1:10,000) and calnexin (Cat#C4731; 1:2000) were obtained from Sigma-Aldrich. Anti-GFP (GF200, Cat#04363-66; 1:10,000) was from Nacalai Tesque (Kyoto, Japan). Anti-cGAS (D1D3G, Cat#15102; 1:1000), anti-DDX41 (D3F1Z, Cat#15076; 1:1000), anti-STING (D2P2F, Cat#13647; 1:1000), anti-MAVS (Cat#3993; 1:1000), and anti-TFAM (D5C8, Cat#8076; 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-influenza virus NS1 (NS1-23-1, Cat#sc-130568; 1:1000), anti-HA (F-7, Cat#sc-7392; 1:1000), anti-myc (9E10, Cat#sc-40; 1:1000), anti-tubulin (DM1A, Cat#sc-32293; 1:2000), anti-Tom20 (FL-145, Cat#sc-11415; 1:1000), anti-Mfn2 (XX-1, Cat#sc-100560; 1:1000), and anti-connexin 43 (F-7, Cat#sc-271837; 1:1000) were purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody against RIG-I (Alme-1, Cat#AG-20B-0009-C100; 1:1000) was from AdipoGen.
Anti ha f 7
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-HA (F-7) is a mouse monoclonal antibody that recognizes the haemagglutinin (HA) epitope tag. It is designed for the detection of proteins that have been engineered to express the HA tag.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2, by Merck Group (6 mentions)
Anti myc 9e10, by Santa Cruz Biotechnology (5 mentions)
Anti gfp gf200, by Nacalai Tesque (1 mentions)
Anti mavs, by Cell Signaling Technology (1 mentions)
Anti sting d2p2f, by Cell Signaling Technology (1 mentions)
Anti cgas d1d3g, by Cell Signaling Technology (1 mentions)
Anti dsdna 35i9 dna, by Abcam (1 mentions)
Anti tubulin dm1a, by Santa Cruz Biotechnology (1 mentions)
Anti tom20 fl 145, by Santa Cruz Biotechnology (1 mentions)
Anti mfn2 xx 1, by Santa Cruz Biotechnology (1 mentions)
Anti lexa, by Santa Cruz Biotechnology (1 mentions)
Anti actin a2066, by Merck Group (1 mentions)
Anti myc n 262, by Santa Cruz Biotechnology (1 mentions)
Anti mtbp k20, by Santa Cruz Biotechnology (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Image studio light, by LI COR (1 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Anti gfp b 2, by Santa Cruz Biotechnology (1 mentions)
Anti gst b 14, by Santa Cruz Biotechnology (1 mentions)
17 protocols using anti ha f 7
1
Antibody Panel for Viral Protein Detection
2
Yeast Two-Hybrid Assay for Protein Interactions
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Y2H assays were performed using the haploid L40ΔG yeast strain co-transformed with LexA-bait and Gal4AD-prey plasmids, selecting for expression of the HIS3 reporter gene (growth on medium lacking leucine and tryptophan (-LW) to select for both plasmids and lacking histidine (-H) to select for HIS3 expression; -LWH medium) (21 (link),24 (link)) and including various concentrations of 3-aminotriazole (3-AT) that increases the stringency of the HIS3 activation, allowing an assessment of the strength of interaction. Western blotting of bait and prey-fusions using anti-HA (F-7) and anti-LexA (2 (link)–12 (link)) antibodies (Santa Cruz Biotechnology) showed stable expression of the fusion proteins, regardless of the Y2H interaction phenotype observed.
3
Quantifying TREX Components in Yeast
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To analyze the global levels of the TREX components, yeast strains expressing myc/HA epitope-tagged TREX components were grown in 25 mL YPD up to an OD600 of 1.0. Yeast cells were then harvested, lysed, and sonicated to prepare the whole-cell extract with solubilized chromatin following the protocol as described previously for the ChIP assay (Bhaumik and Green 2002 (link), 2003 (link); Bhaumik et al. 2004 (link); Shukla et al. 2006 (link)). The whole-cell extract was run on SDS–polyacrylamide gel, and then analyzed by Western blot assay. An anti-myc (9E10; Santa Cruz Biotechnology, Inc.), anti-HA (F-7; Santa Cruz Biotechnology, Inc.), or anti-actin (A2066; Sigma) antibody was used for Western blot analysis.
4
ChIP-qPCR Analysis of MTBP and MYC Interactions
5
Quantitative Protein Tag Detection
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A serial dilution of MBP fused to FLAG®-, HA-, myc- and ALFA-tags was prepared in PBS pH7.4, 0.1 µg mL−1 BSA. In total 1 µL of each dilution was spotted on nitrocellulose membranes. Membranes were blocked and washed with 5% milk powder in TBS-T. Established monoclonal antibodies (anti-FLAG® M2, Sigma #F1804; anti-myc 9E10 Synaptic Systems #343 011; anti-HA F-7, SantaCruz #sc-7392) were used in combination with a secondary goat anti-Mouse IgG IRDye800CW (Li-COR #925–32210, dilution 1:500) to detect FLAG®-, myc- and HA-tag, respectively. The ALFA-tag was detected using a FluoTag®-X2 anti-ALFA nanobody (NanoTag Biotechnologies #N1502) directly coupled to IRDye800CW. All primary antibodies and the nanobody were used at 2.7 nM final concentration. Detection of MBP by a rabbit polyclonal serum recognizing MBP (Synaptic Systems, dilution 1:500) and an anti-rabbit IgG IRDye680RD (Li-COR #925–68071, dilution 1:5000) served as an internal loading control. Membranes were scanned using Odyssey CLx (Li-COR). Quantifications were performed using ImageStudioLight (Li-COR).
6
Yeast Protein Expression and Detection
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Yeast cells harboring BY4741 FBP1-3×FLAG, GID12-6×HA; BY4741 FBP1-3×FLAG, Δgid12; BY4741 FBP1-3×FLAG, pADH::GID12-6×HA; BY4741 MDH2-3×FLAG, GID12-6×HA; BY4741 MDH2-3×FLAG, Δgid12; BY4741 MDH2-3×FLAG, pADH::GID12-6×HA; BY4741 PCK1-3×FLAG, GID12-6×HA; BY4741 PCK1-3×FLAG, Δgid12; BY4741 PCK1-3×FLAG, pADH::GID12-6×HA; BY4741 ICL1-3×FLAG, GID12-6×HA; BY4741 ICL1-3×FLAG, Δgid12; BY4741 ICL1-3×FLAG, pADH::GID12-6×HA were grown in YPD/YPE medium as mentioned in the section “Yeast strains and growth conditions”. 1.0 OD600 equivalent of yeast cells were harvested and flash frozen in liquid nitrogen after overnight non-fermentable carbon source starvation and at indicated time points during carbon recovery.
Cells were lysed using alkaline lysis (2 M NaOH and 7.5% (v/v) 2-mercaptoethanol for 10 min on ice) followed by trichloroacetic acid precipitation (to a final concentration of 15% for 10 min on ice). Proteins were pelleted by centrifugation at 51,428×g for 10 min, and solubilized in 20 µl 2.5× SDS sample loading buffer at 95 °C for 10 min. Samples were loaded on 12% SDS-PAGE gels and then analyzed by western blotting with 1:5000 diluted anti-FLAG M2 monoclonal (Sigma, F1804), 1:5000 diluted anti-HA (F-7) (Santa Cruz Biotechnology, sc-7392), and 1:50,000 diluted anti-Pgk1 (22C5D8) (Invitrogen, Catalog #459250) antibodies (Fig.7d ). Image was taken with classical film development.
Cells were lysed using alkaline lysis (2 M NaOH and 7.5% (v/v) 2-mercaptoethanol for 10 min on ice) followed by trichloroacetic acid precipitation (to a final concentration of 15% for 10 min on ice). Proteins were pelleted by centrifugation at 51,428×g for 10 min, and solubilized in 20 µl 2.5× SDS sample loading buffer at 95 °C for 10 min. Samples were loaded on 12% SDS-PAGE gels and then analyzed by western blotting with 1:5000 diluted anti-FLAG M2 monoclonal (Sigma, F1804), 1:5000 diluted anti-HA (F-7) (Santa Cruz Biotechnology, sc-7392), and 1:50,000 diluted anti-Pgk1 (22C5D8) (Invitrogen, Catalog #459250) antibodies (Fig.
7
Immunoblotting and Immunoprecipitation Protocols
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For immunoblotting, the following antibodies were used as indicated: anti-GST B-14 (Santa Cruz, sc-138) 1:1000 dilution; anti-GFP B-2 (Santa Cruz, sc-9996) 1:1000 dilution; anti-HA F-7 (Santa Cruz, sc-7392) 1:1000 dilution; anti-myc 9E10 (Santa Cruz, sc-40) 1:1000 dilution. For immunoprecipitation, anti-HA 3F10 (Roche, No. 11867431001) 4 μg ml-1 was used.
8
Molecular Tools for CREPT and STAT3 Studies
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Flag-CREPT, Myc-CREPT, Myc-CREPT/RPR, Myc-CREPT/CCT, Flag-STAT3, APRE (acute phase response element)-luciferase and pCDH-HA-CREPT were constructed in this laboratory. HA-p300 plasmid was a gift from Dr. Y. Eugene Chin (Institutes of Biology and Medical Sciences, Soochow University). Anti-STAT3 (c-20), anti-Myc (9E10) and anti-HA (F-7) were purchased from Santa Cruz Biotechnology (Santa Cruz). Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology. Anti-actin (AC-15) and anti-Flag (M2) antibodies were purchased from Sigma. Anti-CREPT antibody was prepared by this laboratory.50 (link) The cytokine leukaemia inhibitory factor (LIF) was purchased from Millipore (cat. #LIF1010). Short interfering RNAs (siRNAs) against CREPT or p300 were synthesised from GenePharma (SuZhou GenePharma Co. Ltd) with the oligo sequence information as shown in Table S1 . The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)-mediated CREPT deletion plasmids were generated based on PX458M vector with guider RNAs and the sequence information was shown in Table S1 .
9
Evaluating Eco1 Mutants' Smc3 Acetylation
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For the spot assays pMET-eco1AID (Y4766) cells were transformed with an empty URA3 plasmid or a URA3 plasmid carrying different versions of Eco1 expressed under Eco1 endogenous promoter. Serial dilutions of stationary cultures were dropped onto YNB-MET-URA as well as YPD + IAA plates and grown for 2 days at 30 °C. To determine the in vivo acetylation of Smc3 by mutant Eco1, cells were grown in YNB CSM-MET and arrested in G1 by addition of alpha factor 1 H. Prior to release, cells were transferred to YPD + IAA + alpha factor. Upon G1 arrest, cells were released into nocodazole for 90 minutes, when proteins were TCA extracted. 15 μg of proteins were separated on 10% SDS-PAGE, transferred and submitted to Western blotting with anti-Acetyl Smc3 (K. Shirahige) and anti HA (F7, Santa Cruz Biotechnology).
10
Flow Cytometry Analysis of BTN3A Chimeras
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293T and 3KO transductants of BTN3As (WT and Chimaeras) were acquired by FACScalibur (BD) and analyzed with FlowJo. For total staining, cells were fixed with fixation buffer for 30 mins at RT, followed by wash and incubated for 30 mins with permeabilization buffer at RT. Then cells were stained with antibodies that were prediluted in permeabilization for 30 mins at 4°C, as per the manufacturer’s instructions (eBiosciences, eBiosciences™ Intracellular Fixation & Permeabilization buffer set). For surface staining, cells were directly stained with antibodies of interest for 30 minutes at 4°C. The BTN3As were detected by unconjugated mAb 103.2 (gift from Daniel Olive). If tagged, unconjugated anti-FLAG (M2, SIGMA) and anti-HA (F-7, Santa Cruz) antibodies were used. The primary antibodies were detected by Fab Donkey anti mouse IgG (H + L)-APC (Jackson Immunoresearch, 115–136-146). mIgG1k and mIgG2a k (eBiosciences) were used as isotype controls.
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