- MA1-19332 - Invitrogen Antibodies SYK antibody

Submitted references Amyloid-β activates NLRP3 inflammasomes by affecting microglial immunometabolism through the Syk-AMPK pathway.

Jung ES, Suh K, Han J, Kim H, Kang HS, Choi WS, Mook-Jung I
Aging cell 2022 May;21(5):e13623

An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes.

Hernandez AA, Foster GA, Soderberg SR, Fernandez A, Reynolds MB, Orser MK, Bailey KA, Rogers JH, Singh GD, Wu H, Passerini AG, Simon SI
Journal of immunology (Baltimore, Md. : 1950) 2020 Nov 15;205(10):2806-2820

CARD9 negatively regulates NLRP3-induced IL-1β production on Salmonella infection of macrophages.

Pereira M, Tourlomousis P, Wright J, P Monie T, Bryant CE
Nature communications 2016 Sep 27;7:12874

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Ramos (Lane 1), THP-1 (Lane 2), RAW 264.7 (Lane 3), Jurkat (Lane 4) and A-431 (Lane 5). The blot was probed with Anti-Syk Monoclonal Antibody (Product # MA1-19332, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 72 kDa band corresponding to Syk was observed in relevant cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blotting analysis of human Syk using mouse monoclonal antibody SYK-01 on lysates of Ramos and Jurkat (negative control) cells under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse anti-Syk Monoclonal antibody (Product # MA1-19332) followed by IRDye800-conjugated anti-mouse secondary antibody. Syk was detected around 72kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blotting analysis of human Syk using mouse monoclonal antibody SYK-01 on lysates of Ramos and Jurkat (negative control) cells under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse anti-Syk Monoclonal antibody (Product # MA1-19332) followed by IRDye800-conjugated anti-mouse secondary antibody. Syk was detected around 72kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of SYK was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR819133_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of SYK was performed by loading 30 µg of A-431 wild type (Lane 1), A-431 CAS9 (Lane 2), A-431 SYK KO (Lane 3) whole cell extracts. The blot was probed with Anti-Syk Monoclonal Antibody (SYK-01)(Product # MA1-19332) using 1:1000 dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to SYK. An uncharacterized band was observed at ~80kDa and ~120kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of SYK was performed by loading 30 µg of WT (lane 1) and SYK CRISPR KO (lane 2) THP-1 cell lysates in RIPA buffer onto a 5-16% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. SYK was detected at approximately 72 kDa using a SYK monoclonal antibody (Product # MA1-19332) at a dilution of 1:1,000 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. The peroxidase-conjugated secondary antibody (Product # 62-6520) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SYK was performed using 70% confluent log phase Ramos cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with SYK Mouse Monoclonal Antibody(Product # MA1-19332) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Syk in human HeLa cell line using anti-Syk (SYK-01; green) Monoclonal antibody (Product # MA1-19332). Actin cytoskeleton was decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Syk in human primary fibroblasts using anti-Syk (SYK-01; green) Monoclonal antibody (Product # MA1-19332). Actin cytoskeleton was decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence of SYK was performed using THP-1 wild-type and SYK KO cells that were transfected with a green or a far-red fluorescent dye, respectively. Post-transfection, WT and KO cells were mixed and plated to a 1:1 ratio on coverslips as a mosaic and incubated for 24 hrs. Cells were fixed in 4% PFA (in PBS) or methanol for 15 min; cells were permeabilized with 0.1% Triton X-100 for 10 min at RT and blocked with PBS with 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 min. Cells were stained with the SYK monoclonal antibody (Product # MA1-19332) at a 1:1,000 dilution overnight at 4°C. Secondary antibody incubation was performed using 1 µg/mL of Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 antibody (Product # A21424) together with DAPI for 1 hr. Imaging was performed with a 40X oil objective and analysis was performed using Image J. Cell image represents a single focal plane; WT and KO cells are outlined with a yellow (WT) or magenta (KO) dashed line. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 CARD9 and SYK regulates NLRP3 activation upstream to speck formation. ( a ) CARD9 and ASC were immuno-labelled in WT, Card9 -/- and Pycard -/- LPS-primed macrophages after nigericin stimulation (5 muM, 30 min). Green arrows indicate CARD9 aggregates. Red arrows indicate ASC specks. ( b ) Co-immunoprecipitation of ASC, CARD9 and SYK from cell lysates of uninfected, S . Typhimurium-infected (m.o.i. 10, 30 min) or nigericin-stimulated (10 muM, 30 min) LPS-primed BMDMs. ( c ) Immunoblot analysis of LPS-primed WT and Card9 -/- uninfected, S . Typhimurium-infected (m.o.i. 10, 2 h) or nigericin-stimulated (10 muM, 30 min) BMDMs. ( a - c ) Images are representative of three independent experiments. Scale bar, 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 4 FIGURE AMPK activation restores Abeta42-induced mitochondrial hyperfission and inflammasome activation in LPS-primed microglia. LPS-primed microglia were pretreated with AICAR (1 mM) for 1 h and then treated with Abeta42 for 24 h. (a-e) Representative immunoblot (a) and densitometric quantifications (b-e) of the protein levels of pAMPK, AMPK, pSyk, Syk, Drp1, pro-IL-1beta, and beta-actin in total cell lysate. (f-g) MitoSOX (red) and DAPI (blue) staining and quantification of MitoSOX fluorescence intensity. Scale bar: 10 mum. (h) Immunostaining of ASC (red) and DAPI (blue). Scale bar: 10 mum. (i) Percentages of microglia containing ASC foci were counted. Data are shown as mean +- SEM from at least three independent experiments and are analyzed by one-way ANOVA and Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

MA1-19332

Antibody data