anti-phospho-stat1 (py701)-pe 58d6  (Cell Signaling Technology Inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: An atlas of protein phosphorylation dynamics during interferon signaling.

doi: 10.1073/pnas.2412990122

Figure Lengend Snippet: Fig. 4. Phosphorylation of PLEKHG3 at S1081 can be mediated by an IFN-regulated kinase in vitro and is required for interaction with 14-3-3 proteins. (A) Phosphopeptide intensities of the PLEKHG3 pS1081 phosphopeptide after stimulation with IFNα2, IFNβ, or IFNω for 90 min from the original LC-MS/MS data. Results from the five independent experiments in the screen are shown, and bars represent mean values with SD. Significance was determined by one-way ANOVA (*P ≤ 0.05). (B) Depiction of a consensus 14-3-3 binding motif and the corresponding sequence in PLEKHG3 surrounding the phosphorylation site at S1081. pS represents phosphorylated serine, and X represents any amino acid. (C and D) HEK293T cells were transiently transfected with 3xFlag-PLEKHG3 WT or the S1081A mutant. PLEKHG3 was then immunoprecipitated from lysates using an antibody against the Flag tag. Immunocomplexes were treated with λ-phosphatase where indicated, and subjected to an in vitro KA with recombinant Flag-IKKε (D). Immunoprecipitates (IP) and total lysates (TL) were analyzed by western blotting with the indicated antibodies. Data shown are representative of at least n = 3 independent experiments. (E–G) HEK293T cells were transiently transfected with 3xFlag-PLEKHG3 WT or the S1081A mutant, together with HA-tagged 14-3-3 proteins or GFP as indicated. 14-3-3 proteins or GFP were immunoprecipitated from lysates using an antibody against the HA-tag. IP and TL were analyzed by western blotting with the indicated antibodies. PLEKHG3 band intensities in IP in (E) were quantified using Image Studio Lite and are shown in (F), where bars represent mean values and SD from n = 3 independent experiments (each dot corresponds to one replicate). Significance compared with 14-3-3β was determined by the one-sample t test on log-transformed data (only significant comparisons are shown: *P ≤ 0.05; **P ≤ 0.01). (H and I) A549 cells were transfected with negative-control siRNAs, an siRNA targeting IRF9, or siRNA pools targeting all seven 14-3-3 family members (14-3-3β, 14-3-3γ, 14-3-3ε, 14-3-3ζ, 14-3-3η, 14-3-3θ, 14-3-3σ). 48 h post transfection, cells were stimulated with IFNα2 (1 ng/mL) for 4 h. Cells were harvested and IFIT2 mRNA levels were determined by RT-qPCR (H). Parallel cells were infected with VSV-GFP (MOI = 1 PFU/cell) and GFP expression as a measure for viral replication was assessed at 24 hpi (I). Bars represent mean values and SD from n = 3 independent experiments (each dot corresponds to one replicate). Significance was determined by two-way ANOVA with Dunnett’s multiple comparison test on log-transformed data (**P ≤ 0.01; ***P ≤ 0.001; ns, nonsignificant). (J) Schematic model for the hypothetical role of the PLEKHG3:14-3-3 axis in IFN signaling: We suggest that IFN-activated IKKε phosphorylates PLEKHG3 and facilitates the interaction of PLEKHG3 with one or more 14-3-3 proteins to regulate signal transduction downstream of STAT1/STAT2 phosphorylation.

Article Snippet: Primary antibodies used were rabbit anti- STAT1- pY701 (#9167, Cell Signaling); mouse anti- STAT1 (sc- 417, Santa Cruz); mouse anti- beta- actin (C4) (sc- 47778, Santa Cruz); mouse anti- ANXA2- pY24 (sc- 135753, Santa Cruz); mouse anti- ANXA2 (sc- 28385, Santa Cruz); rabbit anti- STAT1- pS727 (#9177, Cell Signaling); rabbit anti- STAT1 (#14994, Cell Signaling); mouse anti- MX1 (12), mouse anti- RIG- I (AG- 20B- 0009- C100, Adipogen); rabbit anti- IRF9 (#D2T8M, Cell Signaling); rabbit anti- STAT2- pY690 (#88410, Cell Signaling); mouse antiSTAT2 (A- 7) (sc- 1668, Santa Cruz); rabbit anti- Flag (F7425, Sigma Aldrich); mouse anti- Flag M2 (F1804, Sigma Aldrich); rabbit anti- pSer 14- 3- 3 Binding Motif (14- 3- 3 PBM) (#9601, Cell Signaling); rabbit anti- PLEKHG3 (PA5- 46053, Thermo Fisher Scientific); rabbit anti- HA (#3724, Cell Signaling); and mouse anti- HA (#2367, Cell Signaling).

Techniques: Phospho-proteomics, In Vitro, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Sequencing, Transfection, Mutagenesis, Immunoprecipitation, FLAG-tag, Recombinant, Western Blot, Transformation Assay, Negative Control, Quantitative RT-PCR, Infection, Expressing, Comparison, Transduction