- PA5-28846 - Invitrogen Antibodies SIGLEC8 antibody

Submitted references The Inhibitory Receptor Siglec-8 Interacts With FcεRI and Globally Inhibits Intracellular Signaling in Primary Mast Cells Upon Activation.

Korver W, Wong A, Gebremeskel S, Negri GL, Schanin J, Chang K, Leung J, Benet Z, Luu T, Brock EC, Luehrsen K, Xu A, Youngblood BA
Frontiers in immunology 2022;13:833728

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using SIGLEC8 Polyclonal Antibody (Product # PA5-28846). Sample (30 µg of whole cell lysate). Lane A: A431. 7.5% SDS PAGE. SIGLEC8 antibody. SIGLEC8 Polyclonal Antibody (Product # PA5-28846) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of SIGLEC8 in methanol-fixed HeLa cells using a SIGLEC8 polyclonal antibody (Product # PA5-28846) at a 1:500 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded human breast cancer, using SIGLEC8 (Product # PA5-28846) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Both ITIM motifs are required for Siglec-8 mediated inhibition. (A) Schematic of the Siglec-8 receptor with its two tyrosine residues in the context of ITIM motifs indicated. (B) Percent of CD63 positive MC in unstimulated (ISO + NS, S8 + NS), stimulated (ISO + anti-FcepsilonRI) and inhibited (S8 + anti-FcepsilonRI) MC after transfection of the indicated wild-type or mutant Siglec-8 expression constructs Data are plotted as mean +- SEM (n=3) and are representative of 3 experiments. (C) Percent inhibition of Siglec-8 mAb-treatment for each Siglec-8 expression construct was calculated compared to ISO condition. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA test. (D) Western blot analysis of Siglec-8-Shp2 interaction. Immunoblot (IB) of whole cell lysates (WCL) or FLAG immunoprecipitations (IP) from primary MC mock transfected or with expression constructs for wild-type Siglec-8-FLAG or ITIM double mutant Siglec-8-FLAG. Cells were subjected to PVD treatment for two min and WCL were analyzed for presence of Shp-2 (panel 1) and p-Tyr (panel 4). Anti-FLAG IPs were analyzed for presence of Shp-2 (panel 2), Siglec-8-FLAG (panel 3) and p-Tyr (panel 5). Experiments were performed 2-3 times for confirmation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Siglec-8 interacts with FcepsilonRI machinery components. (A) Western blot analysis of Siglec-8-FcepsilonRIgamma interaction. Immunoblot (IB) of whole cell lysates (WCL) or HA immunoprecipitations (IP) from primary MC transfected with expression constructs for Siglec-8-FLAG (lane 1) or FcepsilonRIgamma-HA (lane 3) or both (lane 2). Blots were developed with anti-FLAG (left panel) and anti-HA (right panel). (B) Western blot analysis of Siglec-8-Lyn interaction. WCL, isotype IP or S8 IP from S8-BMMC were developed using anti-Lyn or anti-Siglec-8 antibodies. (C) Confocal microscopy imaging of Siglec-8 (red) and FcepsilonRI (green) in S8-BMMC. (D) Quantification of confocal images. Left panel: number of FcepsilonRI punctae per cell (PE channel). Right panel: Siglec-8 median fluorescence intensity within FcepsilonRI/Siglec-8 punctae (AF647 channel). Experiments were performed 2-3 times. NS, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA test.

PA5-28846

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