Antibody data
- Antibody Data
- Antigen structure
- References [1]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
- Other assay [1]
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- Product number
- PA5-28584 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- SPHK1 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: 293T, A431, H1299, HeLaS3, HepG2, Molt-4, Raji. Predicted reactivity: Human (99%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 1 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
Submitted references Mfsd2a and Spns2 are essential for sphingosine-1-phosphate transport in the formation and maintenance of the blood-brain barrier.
Wang Z, Zheng Y, Wang F, Zhong J, Zhao T, Xie Q, Zhu T, Ma F, Tang Q, Zhou B, Zhu J
Science advances 2020 May;6(22):eaay8627
Science advances 2020 May;6(22):eaay8627
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using SPHK1 Polyclonal Antibody (Product # PA5-28584). Sample (30 µg whole cell lysate). A: Hep G2. 7.5% SDS PAGE. SPHK1 Polyclonal Antibody (Product # PA5-28584) diluted at 1:1,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of SPHK1 was achieved by transfecting Hep G2 with SPHK1 specific siRNAs (Silencer® select Product # S16959). Western blot analysis (Fig. a) was performed using membrane enriched cell extracts from the knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with SPHK1 Polyclonal Antibody (Product # PA5-28584, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 0.25ug/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SPHK1.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-SPHK1 Polyclonal Antibody, (Product # PA5-28584) and 60 and 40 kDa bands corresponding to SPHK1 isoforms were observed in the cell lines tested except in low expressing SH-SY5Y. Nuclear enriched cell extracts (40 µg lysate) of Hep G2 (Lane 1), U-87 MG (Lane 2) and SH-SY5Y (Lane 3) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blots were probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate(Product # 34076).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of SPHK1 in paraformaldehyde-fixed HeLa cells using a SPHK1 polyclonal antibody (Product # PA5-28584) (Green) at a 1:500 dilution. Alpha-tubulin filaments were labeled with Product # PA5-29281 (Red) at a 1:2000.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of paraffin-embedded SW480 xenograft, using SPHK1 (Product # PA5-28584) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Fig. 3 The S1P signaling pathway is crucial for the maintenance of the BBB. Knock down of S1P1 in the hippocampus led to BBB breakdown. ( A ) Strategy used for S1P1-RNAi transfection. ( B ) Immunostaining for S1P1 showed that the expression of S1P1 was inhibited after RNAi transfection. ( C ) Western blot showing that S1P1 knockdown had no substantial effect on the expression of Sphk1 at D28. ( D ) S1P or PBS was added in situ at 23 days post AAV injection; Evans blue leakage was confined to the hippocampus (black arrowheads). ( E ) Quantification of Evans blue staining showed that additional S1P had no effect on BBB breakdown. ( F ) The inhibition of S1P1 was not affected by additional PBS or S1P. ( G ) The tracer penetrated into the brain parenchyma, indicating that the BBB did not recover after adding S1P. Scale bars: 200 mum in B and F; 100 mum in G. Error bars: SEM. Significance determined by Students t -test: *** P < 0.001, n.s. P > 0.05. Each image is representative of three individual male mice. Photo credit: Fan Wang, State Key Laboratory of Medical Neurobiology, the Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Shanghai Medical College, Fudan University.