PA5-29690

antibody from Invitrogen Antibodies

Targeting: YWHAG 14-3-3GAMMA, 14-3-3γ, PPP1R170

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Recommended by provider)

Immunohistochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-29690

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. Predicted reactivity: Mouse (100%), Rat (100%), Xenopus laevis (98%), Chicken (99%), Rhesus Monkey (100%), Bovine (100%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

1.05 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

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

Western blot

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.

Supportive validation

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.

Supportive validation

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.

Supportive validation

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.

Immunocytochemistry

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.

Immunohistochemistry

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.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

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

Supportive validation

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.