Anti ha 3724

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-HA (3724) is a lab equipment product offered by Cell Signaling Technology. It is a primary antibody that specifically binds to the HA (Hemagglutinin) tag, which is a commonly used epitope tag for protein expression and detection.

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Lab products found in correlation

Anti s6k, by Cell Signaling Technology (2 mentions) Anti caspase 1 p20 14 9832 82, by Thermo Fisher Scientific (2 mentions) Anti aim2, by Thermo Fisher Scientific (2 mentions) Anti phospho p62 s403 mabc186, by Merck Group (2 mentions) Anti lamp 1, by BioLegend (2 mentions) Anti erp72, by Cell Signaling Technology (2 mentions) Anti ha, by Roche (2 mentions) Anti flag, by Cell Signaling Technology (2 mentions) Anti β actin, by Cell Signaling Technology (2 mentions) Anti flag m2, by Merck Group (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Anti flag 14793s, by Cell Signaling Technology (1 mentions) Nbp2 33422, by Novus Biologicals (1 mentions) Irdye 800cw goat anti rabbit igg h l, by LI COR (1 mentions) Anti gapdh 10494 1 ap, by Proteintech (1 mentions) Anti flag, by GenScript (1 mentions) A11012, by Cell Signaling Technology (1 mentions) Alexa fluor 488 goat anti mouse, by Cell Signaling Technology (1 mentions) Sds polyacrylamide gel, by Bio-Rad (1 mentions)

14 protocols using anti ha 3724

Colocalization of CD2v and CD58 in Cells

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PK15 cells were cotransfected with pCD2v-HA and swine pCD58-Flag, fixed with 4% PFA 24 h pt, permeabilized with 0.2% Tritron-X 100 and treated with anti-HA (3724S; Cell Signaling Technology, MA, USA) or anti-Flag (A00187; Genscript, Piscataway, NJ, USA) primary antibodies. Cells were then washed with PBS and incubated with goat anti-rabbit Alexa fluor 594 (A11012; Cell signaling Technology, Danvers, MA, USA) and goat anti-mouse Alexa fluor 488 (A11029; Cell Signaling Technology, Danvers, MA, USA) secondary antibodies for 1 h. Nuclei were stained with DAPI and images obtained using the A1 Nikon confocal microscope. Colocalization of CD2v with endogenous human CD58 was investigated in 293T cells. Cells were transfected with pCD2v-HA and IFA was performed using anti-HA (3724S; Cell Signaling Technology, Danvers, MA, USA) and anti-CD58 (sc20009; Santa Cruz, CA, USA) primary antibodies, and goat anti-rabbit Alexa fluor 594 (A11012; Cell Signaling Technology, MA, USA) and goat anti-mouse Alexa fluor 488 (A11029) secondary antibodies.

Chaulagain S., Delhon G.A., Khatiwada S, & Rock D.L. (2021). African Swine Fever Virus CD2v Protein Induces β-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells. Viruses, 13(8), 1480.

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Antibody Purchase and Production

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Anti-Flag (14793S) and anti-HA (3724S) were purchased from Cell Signaling Technology. Anti-GAPDH (10494-1-AP) was purchased from Proteintech. The rabbit anti-GSDMDC1 polyclonal antibody (NBP2-33422) was purchased from Novus. IRDye 800CW goat anti-mouse IgG (H + L) (926-32210) was purchased from Sera Care, and IRDye 800CW goat anti-rabbit IgG (H + L) (925-32211) was purchased from LI-COR. Polyclonal antibodies against ASFV pS273R, p72, and p30 were prepared by immunizing mice using the purified recombinant ASFV pS273R, p72, and p30 proteins as immunogens. All animal procedures were approved by Harbin Veterinary Research Institute Animal Ethics Committee of CAAS.

Zhao G., Li T., Liu X., Zhang T., Zhang Z., Kang L., Song J., Zhou S., Chen X., Wang X., Li J., Huang L., Li C., Bu Z., Zheng J, & Weng C. (2021). African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D. The Journal of Biological Chemistry, 298(1), 101480.

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NF-kB Reporter Assay in HEK293 Cells

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HEK293 cells were seeded at density of 1.0 × 10 5 cells/mL per well in a 24-well plate and then transfected with pGL4.32-NF-kB-RE (E849A, Promega) luciferase reporter plasmid together with indicated pcDNA3-HA-TAB2 expression vectors using Lipofectamine LTX (Thermo-Fisher Scientific) according to the manufacturer's instructions. Renilla luciferase-expressing plasmid (pRL-SV40, Promega) was used to normalize transfection efficiencies. After 48 hours of transfection, cells were lysed in the passive buffer and assayed for both firefly and Renilla luciferase activities using the Dual-GLO Luciferase Assay System (Promega) in a Glomax 96-Well Microplate Luminometer (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Values are presented as the mean ± standard error of 3 experimental replicates from 3 independent transfections. To verify whether the cells ectopically expressed the wildtype and all aberrant TAB2, lysates from transfected cells were resolved by electrophoresis on 10% sodium dodecylsulfate (SDS) polyacrylamide gel (Biorad), transferred to a nitrocellulose membrane, blocked in gelatin, and then blotted with anti-HA (3724S, Cell Signaling).

Micale L., Morlino S., Carbone A., Carissimo A., Nardella G., Fusco C., Palumbo O., Schirizzi A., Russo F., Mazzoccoli G., Breckpot J., De Luca C., Ferraris A., Giunta C., Grammatico P., Haanpää M.K., Mancano G., Forzano G., Cacchiarelli D., Van Esch H., Callewaert B., Rohrbach M, & Castori M. (2022). Loss-of-function variants in exon 4 of TAB2 cause a recognizable multisystem disorder with cardiovascular, facial, cutaneous, and musculoskeletal involvement. Genetics in medicine : official journal of the American College of Medical Genetics, 24(2).

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Protein-Protein Interaction Analysis

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The overexpression plasmids pCMV-HA-PU.1 and pCMV-Myc-KLF7 were transfected into ICP1 cells. At 48 h after transfection, cells were lysed on ice for 10 min with IP lysis buffer containing 1% protease inhibitor cocktail (Beyotime), and then incubated with 20 μL A/G Plus agarose beads and primary antibodies (anti-HA; #3724; dilution ratio 1:50; Cell Signaling Technology, Danvers, USA; and anti-c-Myc; AM926; dilution ratio 1:100; Beyotime) at 4°C for 3–4 h. After wash with IP lysis buffer, the precipitate was subject to western blot analysis. The experiments were independently repeated three times.

Tan M., Xu H., Li J., Jia Z., Zhang X., Shao S., Zhang W., Wang W, & Sun Y. (2023). PU.1 interacts with KLF7 to suppress differentiation and promote proliferation in chicken preadipocytes: Role of PU.1 and KLF7 in chicken preadipocytes. Acta Biochimica et Biophysica Sinica, 55(1), 143-153.

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Immunodetection of UBE2O and Mxi1 Proteins

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Anti-UBE2O antibody (GTX119315) for immunohistochemical (IHC) analysis was purchased from GeneTex. Anti-UBE2O antibody (A301-873A) for immunoblotting was purchased from Bethyl Laboratories. Anti-Mxi1 antibody (HPA035319), anti-Flag antibody (F1804), and Cycloheximide (01810) were purchased from Sigma-Aldrich. Anti-Myc (sc-40) and anti-Mxi1 (sc-1042) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH (60004-1-Ig) antibody was obtained from Proteintech. Anti-HA (#3724) and anti-GST (#2624) antibodies were obtained from Cell Signaling Technology. Anti-ATM (A19650) and anti-phospho-ATM-Ser1981 (AP0008) antibodies were obtained from ABclonal. The proteasome inhibitor MG132 (474790) was purchased from Millipore. Arsenic trioxide (ATO) was a clinically available drug which was obtained from Beijing ShuangLu Pharmaceutical Co., Ltd., China (approval number: H20080664).

Huang Y., Yang X., Lu Y., Zhao Y., Meng R., Zhang S., Dong X., Xu S, & Wu G. (2020). UBE2O targets Mxi1 for ubiquitination and degradation to promote lung cancer progression and radioresistance. Cell Death and Differentiation, 28(2), 671-684.

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Immunoprecipitation of Flag, HA, and Ubiquitin

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Cells were cultured in 6-well plates at a density of 5 × 10⁵ cells/well overnight before being transfected with the indicated plasmids. After 36 h, the cells were incubated with 20 μM MG132 for 4 h. Total proteins were extracted using cell lysis buffer containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with anti-Flag M2 affinity gel (A2220, Sigma-Aldrich) at 4°C overnight. After washing the beads three times with cell lysis buffer, the immunoprecipitates were collected for immunoblotting. For HA-tagged protein and Ubiquitin protein immunoprecipitation, the lysates were incubated with 1 mg of an anti-HA (3,724, Cell Signaling Technology) antibody, an anti-ubiquitin antibody (sc-271289, Santa Cruz) or IgG overnight and were then incubated with 30 μL of 50% GammaBind plus Sepharose beads (17-0886-01, GE Healthcare, Sweden) for an additional 4 h. After washing the beads three times with cold lysis buffer, the immunoprecipitated proteins were analyzed using immunoblotting.

Fu Y., Zheng Q., Mao Y., Jiang X., Chen X., Liu P., Lv B., Huang T., Yang J., Cheng Y., Dai X., Dai C., Wang X., Yin Y., Song T., Jin W., Zou C., Chen T., Fu L, & Chen Z. (2020). WNT2-Mediated FZD2 Stabilization Regulates Esophageal Cancer Metastasis via STAT3 Signaling. Frontiers in Oncology, 10, 1168.

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Protein Analysis using Diverse Antibodies

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Primary antibodies used were as follows: Anti-HA (#3724), anti-p-YAP^Ser127 (#13008), anti-LATS1 (#3477), anti-p-LATS1^Ser909 (#9157), and anti-β-Trcp (#4394) were from Cell Signaling Technology. Anti-YAP (WH0010413M1), anti-Flag (F1804), anti-PCNA (05-347), and anti-Siah1 (AV38212) were from Sigma. Anti-p-YAP^Tyr357 (ab62751), anti-YAP (ab52771), and anti-SKP1 (ab76502) were from abcam. Anti-Phosphotyrosine (#05-321) was from Millipore. Anti-FRK (sc-166478), anti-CYR61 (sc-374129), and anti-CTGF (sc-101586) were from Santa Cruz Biotechnology. Anti-LATS2 (A16249), anti-p-LATS1^T1079/LATS2^T1041 (AP0912), anti-β-actin (AC026), and anti-Siah1 (A2494) were from ABclonal. Cycloheximide (CHX) and MG132 were from TargetMol. Puromycin, Protein A/G agarose and verteporfin (VP) were from MedChemExpress. PolyJet was from Signagen. Polybrene was from Sigma.

Wang Y., Wang K., Fu J., Zhang Y., Mao Y., Wang X., Wang X., Yu R, & Zhou X. (2022). FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1. Neuro-Oncology, 24(12), 2107-2120.

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Tet-inducible CerS4 Expression and TGF-β Signaling

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Expression of wild-type and catalytically inactive mutant (H212A/H213A) CerS4 was performed using the tet-inducible vector system as described previously (51 (link), 67 (link)). Expression vectors pBABE-puro (#1764), pMD2.G (#12259), psPAX2 (#12260), TβRI (#19161), and TβRII (#11766) were purchased from Addgene. In TGF-β-responsive luciferase reporter plasmids, six copies of the Smad binding element or its mutant form were cloned upstream of the SV40 promoter in the pGL3 vector.
Anti-CerS1 and anti-CerS4 antibodies were purchased from Exalpha Biologicals. Anti–p-Smad3-Ser^423/425 (C255A9) and anti-HA (3724) antibodies were purchased from Cell Signaling Technology. Anti-V5 tag (ab9116) and anti-TβRI (for flow cytometry; ab31013) antibodies were purchased from Abcam. Anti-Smad6/7 (N19, sc-7004), anti-Shh (H-160, sc-9024), anti-Smo (H-300, sc-13943), anti-TβRI (V-22, sc-398), anti-TβR2 (L-21, sc-400), anti-Smad7, anti-IFT88, anti–Ac-tubulin, anti–caveolin-1 (7C8, sc-53564), anti-clathrin heavy chain (TD.1, sc-12734), anti-calnexin (sc-6465), and anti-Smurf2 (H-50, sc-25511) antibodies were purchased from Santa Cruz Biotechnology. Anti-ceramide antibody (MID 15B4) was purchased from Enzo Life Sciences.

Gencer S., Oleinik N., Kim J., Selvam S.P., De Palma R., Dany M., Nganga R., Thomas R.J., Senkal C.E., Howe P.H, & Ogretmen B. (2017). TGF-β receptor I/II trafficking and signaling at primary cilia are inhibited by ceramide to attenuate cell migration and tumor metastasis. Science signaling, 10(502), eaam7464.

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Antibody Characterization and Protein Quantification

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Antibodies were ordered from the following companies: anti-HIF1α (20960-1-AP, 1:1000), anti-HSF1 (51034-1-AP, 1:1000), anti-RhoC (10632-1-AP, 1:1000), and anti-Myc (60003-2-Ig, 1:2000) were from Proteintech (Chicago, IL,USA); anti-HSF1 phosphorylated at Ser326 antibody (ab76076, 1:2000) was from Abcam (Cambridge, MA, USA); anti-HA (#3724, 1:1000), and anti-HSP90α (#8165, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA); anti-RhoA (ARH04, 1:500) and anti-Rac1 (ARC03, 1:500) were from Cytoskeleton (Denver, CO, USA); anti-RhoB (sc-8048, 1:500) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Cdc42 (BA2442, 1:500) was from Wuhan Boster Biological Technology Co. Ltd. (Wuhan, China); and anti-Flag (F1804, 1:1000) was from Sigma-Aldrich. Cells were washed with PBS three times after treatment. Whole cell lysates were prepared using RIPA lysis buffer (Merck Millipore), with the addition of complete protease inhibitors (Roche). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific) and approximately 20 μg of cell lysates were used. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and exposed in a Tanon-5200 machine.

Guo Y., Wang J., Zhou K., Lv J., Wang L., Gao S., Keller E.T., Zhang Z.S., Wang Q, & Yao Z. (2020). Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(6).

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Immunoblot Antibodies for Inflammasome Research

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For Immunoblot: Cell Signaling Technology: anti-β-Actin (3700), anti-ERP72 (5033), anti-S6K (2708), anti-α-Tubulin (3873), anti-FLAG (14793). eBiosciences: anti-Caspase-1 p20 (14-9832-82), anti-AIM2 (14-6008-93). Santa Cruz Technologies: anti-ASC (sc-22514-R), anti-TGN38 (sc-166594), anti-HistoneH1 (sc393358). Sigma: anti-Caspase-11 (C1354), anti-GasderminD (G7422), anti-FLAGM2 (F3165). Adipogen: anti-NLRP3 (AG-20B-0014-C100), anti-ASC (AG-25B-0006-C100). Millipore: anti-Phospho-p62 S403 (MABC186). Research & Development: anti-IL-1β (AF-401-NA). Roche: anti-HA (11815016001). Abcam: anti-GasderminD (ab219800), anti-NEK7 (ab133514), anti-Calreticulin (ab92516), anti-GM130 (ab52649), anti-CathepsinS (ab232740), anti-LPS (ab35654). Biolegend: anti-LAMP1 (121602). For immunoprecipitation of HA-NLRP3: Cell Signaling: anti-HA (3724).

Moretti J., Jia B., Hutchins Z., Roy S., Yip H., Wu J., Shan M., Jaffrey S.R., Coers J, & Blander J.M. (2022). Caspase-11 Interaction with NLRP3 Potentiates the Noncanonical Activation of the NLRP3 Inflammasome. Nature immunology, 23(5), 705-717.

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