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Western blotAntibody data
- Antibody Data
- Antigen structure
- References [1]
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- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
- Other assay [4]
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- Product number
- PA5-29690 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- 14-3-3 gamma Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: K562, THP-1, HL-60, 293T, A431, H1299, HeLa, HepG2, Molt-4, Raji, mouse brain.
- Concentration
- 1.05 mg/mL
Submitted references PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2.
Civiero L, Cogo S, Kiekens A, Morganti C, Tessari I, Lobbestael E, Baekelandt V, Taymans JM, Chartier-Harlin MC, Franchin C, Arrigoni G, Lewis PA, Piccoli G, Bubacco L, Cookson MR, Pinton P, Greggio E
Frontiers in molecular neuroscience 2017;10:417
Frontiers in molecular neuroscience 2017;10:417
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of 14-3-3 gamma using 30 µg of A) 293T and B) A431 lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a 14-3-3 gamma polyclonal antibody (Product # PA5-29690) at a dilution of 1:1000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts of U-87-MG (Lane 1), HEK 293 (Lane 2), Neuro-2a (Lane 3), A-431 (Lane 4), tissue extract of Mouse Brain (Lane 5) and Rat Brain. The blot was probed with Anti-14-3-3 gamma antibody (Product # PA5-29690, 1µg/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 28 kDa band corresponding to 14-3-3 gamma was observed across the cell lines and tissues tested.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot analysis of 14-3-3-gamma was performed by separating 30 µg of various whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a 14-3-3 gamma Polyclonal Antibody (Product # PA5-29690) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using 14-3-3 gamma Polyclonal Antibody (Product # PA5-29690). Sample (50 µg of whole cell lysate). Lane A: mouse brain. 12% SDS PAGE. 14-3-3 gamma Polyclonal Antibody (Product # PA5-29690) diluted at 1:10,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of 14-3-3 gamma in methanol-fixed HeLa cells using a 14-3-3 gamma polyclonal antibody (Product # PA5-29690) at a 1:200 dilution.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of paraffin-embedded DLD1 xenograft, using 14-3-3 gamma (Product # PA5-29690) antibody at 1:500 dilution. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 2 14-3-3gamma is a PAK6 substrate. (A) Recombinant His-14-3-3 isoforms were incubated with Flag-PAK6 wild-type, K436M and S531N (4:1 molar ratio) in the presence of 33 P-ATP/Mg 2+ for 1 h and subjected to SDS-PAGE, transferred onto nitrocellulose membranes and imaged with a phosphoscreen. Total protein amount was verified by western blot with anti-Flag and anti-His antibodies. (B) Quantification of incorporated 33 P-radioactivity normalized by total 14-3-3 ( n = 4 experiments. Data are presented as mean +- SEM. Two-way ANOVA followed by Tukey's multiple comparison test ( * P
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 3 PAK6-mediated phosphorylation at Ser59 regulates 14-3-3gamma affinity for target proteins. (A) Schematic of 14-3-3 protein sequence and selected phospho-sites identified by LC-MS/MS containing a group II PAK consensus sequence. (B) Recombinant His-14-3-3gamma was incubated with Flag-PAK6 S531N (4:1 molar ratio) in the presence or in the absence of 1 mM ATP and subjected blue native gel separation. (C) Recombinant His-14-3-3 wild-type, S59D and T145D quaternary structure were investigated by blue native gel electrophoresis. (D) Total brain proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and subjected to overlay assays using recombinant 14-3-3gamma wild-type, S59D, T145D, and PAK6-phosphorylated (p14-3-3). Anti-His antibody, anti-betaeta-tubulin and coomassie staining were used to verify bound 14-3-3 and protein loading, respectively. Representative overlay assays and western blots from n = 3 independent replicates. Experiments were run in two technical replicates per each condition and in parallel to allow for comparison.
