- MA1-13042 - Invitrogen Antibodies STAT3 antibody

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Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

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FGF2 regulates melanocytes viability through the STAT3-transactivated PAX3 transcription.

Dong L, Li Y, Cao J, Liu F, Pier E, Chen J, Xu Z, Chen C, Wang RA, Cui R
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of STAT3 using anti-STAT3 (9D8) monoclonal antibody (Product # MA1-13042) (shown in green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a mouse monoclonal antibody recognizing STAT3 (Product # MA1-13042), at a dilution of 1:100 for at least 1 hour at room temperature. Cells were then washed with PBS and incubated with DyLight 488 goat-anti-mouse secondary antibody (Product # 35503) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Signal transducer and activator of transcription 3 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 5 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with STAT3 Monoclonal Antibody (9D8) (Product # MA1-13042) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nucleus and cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60 magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of STAT3 was performed using cross-linked chromatin from 1x10^6 HTC-IR rat hepatoma cells treated with insulin for 0, 10, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a STAT3 monoclonal antibody (Product # MA1-13042). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify exon-1 or exon-28 of the LAMC1 gene. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the rat LAMC1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by LAMC1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of STAT3 was performed on HepG2 cells. The antigen:antibody complex was formed by incubating 750 µg whole cell lysate with 2 µg of mouse monoclonal antibody recognizing STAT3 (Product # MA1-13042) overnight on a rocking platform at 4øC. The immune-complex was then captured on 50 µL Protein A/G Plus Agarose (Product # 20423). Captured immune-complexes were then washed extensively and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing STAT3 (Product # MA1-13042) at a dilution of 1:5000 overnight rotating at 4øC. Membranes were washed in TBST and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:2000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Pierce Super Signal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 5 NEK6 inhibits apoptosis through the activation of STAT3. (A) Immunoblot analysis of the phosphorylated STAT1 (p-STAT1), STAT1, phosphorylated STAT3 (p-STAT3), and STAT3 in RAW 264.7 cells stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis . (B) Immunoblot analysis of the p-STAT1, STAT1, p-STAT3, and STAT3 in RAW 264.7 cells and BMDMs transfected with Myc-NEK6. (C) BMDMs from WT and NEK6-deficient ( Nek6 -/- ) mice were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis for 24 h. The expressions of p-STAT1, STAT1, p-STAT3, STAT3, and NEK6 were analyzed by Western blotting. (D) Immunoblot analysis of the p-STAT3 and STAT3 in lungs, spleens, and livers from WT and Nek6 -/- mice at 7 dpi. (E and G) The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 in 10 mug/mL of gamma-irradiated M. tuberculosis -stimulated BMDMs from WT and Nek6 -/- mice were calculated by qRT-PCR at the indicated times (E) and by Western blotting (G). (F and H) BMDMs from Nek6 -/- mice were pretreated with 20 ng/mL IL-6 for 4 h h, then cells were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis . The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 were calculated by qRT-PCR (F) and Western blotting (H) at the indicated times. (I) BMDMs from WT and Nek6 -/- mice were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis for 24 h, and the apoptosis rates were detected by flow cytometry. (J) BMDMs from Nek6 -/- mice were pretreated with 20 ng/mL of I

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Western blot of STAT3 protein expression levels in LFs treated with anti-miR-21. Quantification of immunoblots using anti-STAT3 of LFs from BAL of CLAD patients treated with anti-miR-21 after 48h. Control cells (CTR); negative control treated cells (NC).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 STAT3 expression in the LCWE-induced model of vasculitis and response to anakinra treatment. (A) Representative images of immunohistochemistry staining for STAT3 in murine heart of PBS control, LCWE injected or LCWE injected mice treated with anakinra. (B) STAT3 staining score in PBS control, LCWE injected and LCWE injected mice treated with anakinra. (C) Representative images of pSTAT3 immunofluorescence staining in murine heart tissue from PBS control and LCWE injected mice. (D) Quantification of pSTAT3 immunofluorescence staining in murine heart tissue. (E) Western blot of pSTAT3 and STAT3 in murine heart tissue from PBS control and LCWE injected mice. (F) Quantification of pSTAT3/STAT3 ratio from Western Blot. Data was analyzed by one-way ANOVA with Tukey''s multiple testing comparison (B) or Student''s t test (D, F) . * p < 0.05, ** p < 0.01, **** p < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Extended Data Figure 1. STAT3 is S-palmitoylated at evolutionarily conserved cysteine residues and mutation of STAT3 palmitoylation sites does not affect disulfide formation and protein stability. (a) HEK293A cells were incubated for 4 hours with DMSO or 50 muM clickable probes. Cell lysates were reacted with azide-biotin for enrichment of labeled proteins with streptavidin beads and identified by mass spectrometry. The peptide spectral counts were shown in the table. ( b ) HEK293A cells were labeled with 50 muM chemical probe (Alk-C16) for 4 hours. Cell lysates were reacted with biotin-azide and precipitated with streptavidin beads and subjected to western blot using anti-STAT3 antibody. Endogenous STAT3 palmitoylation was analyzed by western blot. (c) HEK293A cells were transfected with Myc-STAT3 WT and labeled with 50 muM chemical probe Alk-C16 for 4 hours, cell lysates were subjected to Streptavidin blot following anti-Myc IP and subsequent Click reaction. (d) HEK293A cells were transfected with empty vector or GFP-STAT1 and labeled with 50 muM chemical probe Alk-C16 for 4 hours. After Click reaction, cell lysates were subjected to streptavidin blot showing detection of STAT1. (e) Similar analysis as panel (c) was performed in HEK293A cells transfected with Flag-STAT1alpha and Flag-STAT1beta. (f) HEK293A cells were transfected with Flag-STAT3 or Flag-STAT5 and labeled with 50 muM chemical probe Alk-C16 for 4 hours, Cell lysates were reacted with biotin-azide and precipita

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Extended Data Figure 7. ZDHHC19 mediated STAT3 palmitoylaiton facilitates LSCC tumor cell growth, colony formation and migration in vitro through induction of STAT3 activity. (a) HCC95 lung squamous cell carcinoma cells were pretreated with ruxolitinib (1 muM) for 30 minutes and then labeled with 50 muM probe Alk-C16 for 2 hours with the incubation of IL-6 (20 ng/ml). Endogenous STAT3 palmitoylation was analyzed by Click reaction and streptavidin beads pulldown, and followed by western blotting. (b) HCC95 cells were treated as in panel (a). Whole cell lysates were analyzed by anti-STAT3 IP followed by western blotting using the indicated antibodies. (c) Lung squamous cell carcinoma cell lines HCC95 and KNS-62 were transfected with shRNA control or shZDHHC19 (3 and 5) and labeled with 50 muM Alk-C16 probe for 4 hours, Cell lysates were reacted with biotin-azide and precipitated with streptavidin beads. Endogenous STAT3 palmitoylation was determined by western blot. (d) SK-MES-1 cells were transfected with ZDHHC19-shRNA and labeled with 50 muM palmitoylation probe Alk-C16 for 4hours. Cells lysates were reacted with azide-biotin and subjected to streptavidin beads pull-down. STAT3 palmitoylation was shown by western blot. (e) HCC95 shRNA control cells and Sh3 ZDHHC19 stable cells were pretreated with ruxolitinib (1 muM) for 30 minutes and then labeled with 50 muM probe Alk-C16 for 2 hours with the incubation of IL-6 (20 ng/ml). Endogenous STAT3 palmitoylation was determined by w

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 2 FIGURE Characterization of KAIMRC1 cells on protein level. (A) Western blot analysis of the KAIMRC1 cell line in normal (+) and serum-starved conditions(-) against P- CSF2RB , CSF2RB , P-AKT1, AKT1, JAK2, P-JAK2, STAT3, P-STAT3, STAT5, mTOR, and P-mTOR. Ligand-independent activation of AKT/mTOR pathway, as well as the JAK2/STAT pathway, was observed. (B) Comparative Western blot analysis of KAIMRC1, MDA-MB-231, and MCF-7 cell lines against CSF2RB . (C) Schematic representation of proposed activation of the AKT/mTOR pathway and JAK2/STAT pathway that might result in cell cycle survival and proliferation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 2 Characterization of KAIMRC1 cells on protein level. (A) Western blot analysis of the KAIMRC1 cell line in normal (+) and serum-starved conditions(-) against P- CSF2RB , CSF2RB , P-AKT1, AKT1, JAK2, P-JAK2, STAT3, P-STAT3, STAT5, mTOR, and P-mTOR. Ligand-independent activation of AKT/mTOR pathway, as well as the JAK2/STAT pathway, was observed. (B) Comparative Western blot analysis of KAIMRC1, MDA-MB-231, and MCF-7 cell lines against CSF2RB . (C) Schematic representation of proposed activation of the AKT/mTOR pathway and JAK2/STAT pathway that might result in cell cycle survival and proliferation

MA1-13042

Antibody data