The following antibodies were purchased from eBioscience and used for FACS assay: anti–mouse CD3e (145-2C11), anti–mouse CD4 (GK1.5), anti–mouse CD8a (53–6.7), anti–mouse CD45R (B220; RA3-6B2), anti–mouse IL-17A (eBio17B7), anti–mouse IFN-γ (XMG1.2), anti–mouse CD11b (M1/70), anti–mouse CD29 (eBioHMb1-1), and anti–mouse CXCR4 (2B11). The following antibodies were purchased from BD: anti–mouse-Vα2 (B20.1), anti–mouse-Vα3.2 (553219), anti–mouse-Vβ6 (553194), anti–mouse-Vβ8.1/8.2 (118405), anti–mouse-Vβ8.3 (118603), anti–mouse-Vβ11 (125907), and anti–mouse-Vβ14 (553258). The following antibodies were purchased from to be added: anti–mouse CCR5 (HM-CCR5; BD), anti–mouse CD44 (IM7; eBioscience), and anti–mouse Foxp3 (FJK-16S; eBioscience). Anti–human CD4 (S5.3; Invitrogen), anti–human DOCK8 (sc-104911; Santa Cruz Biotechnology, Inc.), and anti–human LRCH1 (sc-84195; Santa Cruz Biotechnology, Inc.) were used for FACS or immunoblotting assay. Anti-Phosphoserine/threonine/tyrosine antibody (ab15556; Abcam), anti-FLAG (F3165; Sigma-Aldrich), anti-HA (H3663; Sigma-Aldrich), anti-c-Myc (C3956; Sigma-Aldrich), and anti-Cdc42 (sc-87; Santa Cruz Biotechnology, Inc.) were used for immunoprecipitation or immunoblot assay.
Anti ha h3663
Manufactured by Merck Group
Anti-HA (H3663) is a laboratory reagent produced by Merck Group. It serves as an antibody that specifically binds to the hemagglutinin (HA) tag, a commonly used protein tag in molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Sc 9996, by Santa Cruz Biotechnology (2 mentions)
Sc 11049, by Santa Cruz Biotechnology (2 mentions)
Sc 520, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Mes running buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Nupage mops, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 f1804, by Merck Group (2 mentions)
Ab138210, by Abcam (2 mentions)
Anti yap, by Abcepta (2 mentions)
Anti gfp, by Santa Cruz Biotechnology (2 mentions)
Ab6276, by Abcam (2 mentions)
Anti c myc c3956, by Merck Group (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Anti phosphoserine threonine tyrosine antibody, by Abcam (1 mentions)
Anti ubiquitin p4d1, by Santa Cruz Biotechnology (1 mentions)
Prolong gold mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 488, by Jackson ImmunoResearch (1 mentions)
Anti flag f1804, by Merck Group (1 mentions)
8 protocols using anti ha h3663
1
Multicolor Flow Cytometry for Immune Cell Profiling
2
Immunofluorescence Staining of Ubiquitin
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One day prior transfection and/or electroporation cells were seeded on glass coverslips in a 35 mm2 dish. For antibody staining cells were fixed with 4% PFA and washed three times with PBS. Coverslips were blocked for 20 min with PBS containing 2% FBS, 1% BSA and 0.4% Triton-X100. After incubation with primary antibodies anti-HA (H3663, Sigma-Aldrich, 1:200) and anti-ubiquitin P4D1 (Santa Cruz, 1:100, sc-8017) for two hours, coverslips were washed three times with PBS and incubated with secondary antibody (anti-mouse-Alexa488, Jackson Laboratories) for one hour. Lastly, coverslips were washed three times with PBS and mounted on object glasses using ProlongGold mounting medium with DAPI (Life technologies, P36931). All procedures were performed at room temperature.
3
Immunoblotting for Protein Detection
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Equal total protein amounts were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis before transferring to polyvinylidene difluoride membrane via a MiniTrans‐Blot electrophoretic transfer cell (Bio‐Rad). Primary antibodies were then used to probe membranes at the following dilutions: anti‐HA (H3663; Sigma‐Aldrich), 1 : 2000; anti‐GUS (G5420; Sigma‐Aldrich), 1 : 1000. anti‐FLAG (F1804; Sigma‐Aldrich), 1 : 1000; anti‐FIE (AS12 2616; Agrisera, Vännäs, Sweden), 1 : 1000. Horseradish peroxidase‐conjugated anti‐mouse or rabbit secondary antibodies (sc‐358914 and sc‐2004; Santa Cruz, Dallas, TX, USA) were used at a titre of 1 : 10 000, before developing to film using ECL Western blotting substrate (Thermo Fisher Scientific).
4
Antibody Validation and Characterization
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The antibodies used in our study are: anti-HA (H3663, 1:2000 dilution) from Sigma; anti-H3 (ab1791, 1:5000 dilution), anti-H3K9me3 (ab8898, 1:2000 dilution), anti-H3K4me3 (ab8580, 1:2000 dilution), anti-H3K36me2 (ab9049, 1:2000 dilution), anti-H3K36me3 (ab9050, 1:2000 dilution), anti-H4K20me2 (ab9052, 1:2000 dilution) and anti-H4K20me3 (ab9053, 1:2000 dilution) from Abcam; anti-H1 (pAb)(61202, 1:2000 dilution) from Active Motif; anti-H3K27me3 (9733S, 1:2000 dilution) and anti-SUZ12 (3737S, 1:2000 dilution) from CST; anti-H3K9me2 (NB21-1072S, 1:2000 dilution) from NOVUS and anti-H3K4me2 (07-030, 1:2000 dilution) from Millipore; anti-V5 (P01L075, 1:2000 dilution) from Gene-Protein Link. The uncropped and unprocessed scans of all of the blots are shown in the Source Data file.
5
Immunoprecipitation of MyD88 Protein
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For immunoprecipitation cell lysates (RIPA buffer with phosphatase and protease inhibitors) were incubated for 1.5 h with MyD88 D80F5 (CST) or anti-GFP G1544 as control antibody (Sigma), then protein G dynabeads (Thermo Fisher) were added for 1.5 h at 4°C. Beads were washed 3 times with RIPA buffer and the bound proteins were eluted with 2X LDS sample buffer (Thermo Fisher). For immunoblot cell lysates or elutions were separated on 10% or 4%–12% SDS-PAGE gels. Proteins blotted onto nitrocellulose membranes were probed with anti-HA H3663 (Sigma-Aldrich), MyD88 4D6 (epitope surrounding Leu77, Thermo Fisher), MyD88 D80F5 (epitope surrounding Cys233, CST) and MyD88 3699 (epitope surrounding Lys119, CST), HRP-conjugated secondary antibodies (1:8000) or HRP-conjugated anti-Mouse IgG (1:1000, Kappa light chain) and visualized using CCD-based ECL detection. Further details in Supplemental Information
6
Western Blot Analysis of Key Signaling Proteins
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Cells were lysed with RIPA buffer supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Roche). Lysates were denatured by heating for 5 minutes at 95 °C and loaded onto 4–12% Bis-Tris polyacrylamide gel. NuPAGE MOPS or MES running buffer (Invitrogen) was used for the SDS-PAGE. The proteins were subsequently transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked and incubated with primary antibodies and secondary HRP-conjugated antibodies, and developed by exposure to film. Antibody and dilutions used in the studies: anti-FLAG M2 (F1804, Sigma Aldrich, 1:5000), anti-HA (H3663, Sigma Aldrich, 1:1000), anti-GFP (sc-9996, Santa Cruz, 1:2000), anti-YAP (AT4556a, Abgent, 1:1000), anti-pYAP (13008S, Cell signaling, 1:1000), anti-SCRIB (sc-11049, Santa Cruz, 1:3000), anti-ZDHHC7 (ab138210, Abcam, 1:500), anti-AKT (4691S, Cell signaling, 1:1000), anti-pAKT(4060S, Cell signaling, 1:1000), anti-MEK(4694S, Cell signaling, 1:1000), anti-pMEK (9154S, Cell signaling, 1:1000), anti-H-Ras (sc-520, Santa Cruz, 1:1000), anti-GAPDH (2118S, Cell signaling, 1:1000), anti-β-actin (ab6276, Abcam, 1:500). Full images of blots are shown in the supplementary information (Supplementary Figs. 33–49)
7
Expression and Analysis of ALK1 Mutants
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HeLa cells were transfected using Lipofectamine 2000 (Invitrogen) following the supplier’s instructions with 2μg of plasmids encoding WT-ALK1 or different mutants. After 48 hours incubation, cells were washed twice with PBS (Phosphate-Buffered Saline) and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1% Triton X-100) with a cocktail of protease inhibitors (Complete, Roche). Protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and analyzed by immunoblotting with anti-HA (H3663; Sigma- Aldrich) and anti-βactin (A5316; SIGMA) antibodies.
8
Western Blot Analysis of Key Signaling Proteins
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