Antibodies and reagents used for flow cytometry were: CD8-APC, Fixable viability stain 450, antiCD16/32 (eBiosciences); anti-rabbit-PE (Jackson Immunoresearch); anti-SLP76 (pY128), CD4-PE, CD25-FITC, CD8 FITC and anti-mouse IgG1-PE (BD Biosciences); CD4-PEVio770 (Miltenyi Biotec); CD3-BV421 (Biolegend); anti-pErk1/2 (pT202/pY204) (Cell signaling Technology). The following reagents were used for cell stimulation, immunoblotting or immunoprecipitation: anti-CD3-biontinylated, anti-CD28-biontinylated, anti-CD3ε, anti-CD28 (eBiosciences); streptavidin (Sigma); anti-pErk1/2 (pT202/pY204), anti-pPLCγ1 (pY783), anti-pp38 (pT180/pY182), anti-pJNK (pT183/Y185), anti-pAkt (pS473), anti-pSLP76 (pS376) (Cell Signaling Technology); anti-GADS (Santa Cruz Biotechnology Inc.); rabbit anti-SLP76 or goat-anti-SLP76 (Thermo Fisher Scientific); anti-β-tubulin (Chemicon); for anti-14-3-3 immunoblotting we used a mix of anti-pan 14-3-3 and anti-14-3-3ζ (Santa Cruz, Biotechnology Inc.).
Anti pan 14 3 3
Manufactured by Santa Cruz Biotechnology
Anti-pan 14-3-3 is a laboratory reagent that can be used to detect the presence of 14-3-3 proteins, which are a family of highly conserved regulatory proteins found in all eukaryotic cells. This antibody recognizes multiple isoforms of the 14-3-3 protein, allowing for the identification and quantification of total 14-3-3 levels in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti 14 3 3ζ, by Santa Cruz Biotechnology (2 mentions)
Anti taz, by BD (2 mentions)
Anti c myc, by Santa Cruz Biotechnology (2 mentions)
Cd4 pe, by BD (1 mentions)
Cd8 fitc, by BD (1 mentions)
Cd25 fitc, by BD (1 mentions)
Cd8 apc, by Thermo Fisher Scientific (1 mentions)
Cd3 bv421, by BioLegend (1 mentions)
Anti mouse igg1 pe, by BD (1 mentions)
Anti cd16 32, by Thermo Fisher Scientific (1 mentions)
Anti slp76, by BD (1 mentions)
Anti p plcγ1, by Cell Signaling Technology (1 mentions)
Anti perk1 2, by Merck Group (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Cd4 pe vio770, by Miltenyi Biotec (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
Phos tag gel, by Fujifilm (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Okadaic acid, by Merck Group (1 mentions)
Leptomycin b, by Merck Group (1 mentions)
7 protocols using anti pan 14 3 3
1
Comprehensive T-cell Signaling Antibody Panel
2
Western Blot Protein Detection Protocols
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For western blot analysis, proteins were detected using the following antibodies: anti-TAZ (BD Biosciences, 560235, 1:1000), anti-RAN (Cell Signaling Technology, #4462, 1:1000), anti-c-Myc (Santa Cruz Biotechnology, SC-40, 1:1000), anti-pan-14–3–3 (Santa Cruz Biotechnology, SC-629, 1:1000), and anti-GFP (SC-8334, 1:1000). Jackson ImmunoResearch Laboratories was the source for HRP, IRDye680RD, and IRDye800CW conjugated secondary antibodies. Alexa 488 or 555 conjugated secondary antibodies were from Invitrogen. HRP conjugated secondary antibodies were used in 1:5000 for western blot analysis, fluorescent secondary antibodies 1:10000. Leptomycin B, Rapamycin, and okadaic acid were purchased from Sigma Aldrich and RhoII activator from Cytoskeleton Inc. Phos-tag gels (Wako Pure Chemical Industries, Ltd.) were ordered from Cedarlane.
3
Immunodetection of BCR-ABL Fusion Protein
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We generated an anti‐BCR‐ABL (e14a2) junction‐specific Ab for immunocytochemistry by immunizing rabbits with the peptide C + KQSSVPTSSKENLL corresponding to amino acids 78‐91 (EU394718.1) of the e14a2‐type BCR‐ABL protein (Figure S1 ). Specificity of the antibody was validated by peptide blocking and negative staining of e1a2‐type BCR‐ABL protein in Ph‐positive ALL cells (Figure S2 ). For immunofluorescent staining, cells were placed on a glass slide using a Cytospin centrifuge (Shandon Scientific) or cultured on a chamber slide and fixed using an Image‐iT Fixation/Permeabilization Kit (Thermo Fisher Scientific).24 These specimens were stained with a rabbit BCR‐ABL junction‐specific Ab and anti‐p62 (MBL), anti‐CD34, or anti‐pan 14‐3‐3 (Santa Cruz Biotechnology) Abs. We used Alexa Fluor 594‐conjugated anti‐rabbit IgG and Alexa Fluor 488‐conjugated anti‐mouse IgG Abs (Invitrogen) as secondary Abs. Finally, nuclei were counterstained with ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology). Stained samples were observed under a BZ‐X fluorescence microscope (Keyence).
Other conventional techniques and reagents are described in DocumentS1 .
Other conventional techniques and reagents are described in Document
4
Western Blot Antibody Detection Protocol
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For western blot analysis, proteins were detected using antibodies obtained from various commercial sources including anti-TAZ (BD Biosciences, 560,235, 1:1,000) and anti-HA (Covance, MMS-101P, 1:1,000). Antibodies purchased from Santa Cruz Biotechnology included anti-c-Myc (sc-40, 1:1,000) anti-MRTF (sc-47282, 1:500) anti-pan-14-3-3 (sc-629, 1:1,000), anti-GAPDH (sc-47724, 1:20,000), anti-CTGF (sc-14939, 1:1,000) and anti-Cyr61(sc-8561, 1:1,000). Cell Signaling Technology was the source for anti-SMAd3 (#9513, 1:1,000), anti-YAP/TAZ (#8418, 1:1,000) and anti-SRF (#5147, 1:1,000), and Sigma-Aldrich for anti-FLAG/M2 (F1804, 1:1,000) and anti-SMA (AF228; 1:5,000). Anti-histone was from EMD Millipore (MAB052, 1:500) and anti-Filamin A was from Abcam (ab51217, 1:500). Anti-BSAC antibody was a gift from H. Nakano and was described previously64 (link). Normal goat, rabbit and mouse IgG were from Santa Cruz Biotechnology (sc-2028, sc-2027 and sc-2025, respectively). Jackson ImmunoResearch Laboratories was the source for all horseradish peroxidase-conjugated secondary antibodies (1:5,000). TGFβ was purchased from R&D Systems and JK from EMD.
5
HCMV-UL32-GFP Infection in Fibroblasts
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Human fibroblasts infected with HCMV-UL32-GFP were fixed with 4% formaldehyde for 120 min while shaking. After fixation, cells were pelleted, cryoprotected in 2.3 M sucrose and plunge-frozen on pins for Tokuyasu sectioning75 . For immunogold labelling, ultrathin sections were collected on coated grids, blocked and co-stained with anti-GFP (1:250, 132005, Synaptic Systems), anti-pan 14-3-3 (1:50, clone H8, sc-1657, Santa Cruz) and anti-PP-1(1:50, anti-PP-1, clone E9, sc-7482, Santa Cruz) followed by secondary antibodies gp-12 nm gold (species: guinea pig) and ms-18 nm gold (species: mouse). After washing, sections were contrasted and covered with polyvinyl alcohol and tungsto-silicic acid hydrate. Stained ultrathin sections were examined with a Zeiss 902 transmission electron microscope at 80 kV and photographs were taken with a Morada G2 TEM camera.
6
Antibody Characterization for Protein Signaling
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The antibodies used in this work, were as follows: Anti-14-3-3ζ (1:2000), anti-Pan 14-3-3 (1:2000), anti-MEF-2D (1:1000), anti-RBM25 (1:1000), anti-Bcl-x (1:1000), anti-pERK (44/42) (1:1000) and anti-ERK (1:1000) were purchased from Santa Cruz Biotechnology; anti-actin (1:100,000) from Sigma Aldrich; anti-RXRXXpS/T (1:1000) and anti-pS660 PKC β II (1:1000) from cell signaling; anti-pS473-AKT (1:5000) from R&D, anti-Pan-AKT (1:4000) was purchased from Invitrogen and goat F(ab’)2 anti-Human IgM from Southern Biotech. Polyclonal rabbit antibody against BTK-SH3 domain has been described earlier [22 (link)].
7
Ebselen Treatment and 14-3-3 Protein Localization
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In total, 20,000 SHSY5Y cells per well were seeded on coverslips in four-well plates and, 5 hours later, were treated with 5 mM ebselen and DMSO (0.05%). Cells were then incubated for 18 hours before subjecting to immunofluorescence investigation. Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and then permeabilized with PBS 0.2% Triton and blocked with 5% FBS in PBS. Primary antibody rabbit anti-pan-14-3-3 (1:100; Santa Cruz) and mouse anti-b-actin (1:100; Sigma) and secondary antibody antirabbit Alexa Fluor 488 (A-11008, RRID: AB_143165; 1:200; Molecular Probes) and anti-mouse Alexa Fluor 555 (A-21422, RRID: AB_141822; 1:200; Molecular Probes) were used. DAPI (Molecular probes) was used as a nuclear stain. Images were obtained by using Leica microscope TCS SP5 (Leica Microsystem GmbH).
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