- 44-494G - Invitrogen Antibodies KIT antibody

Submitted references Dihydromyricetin ameliorates osteogenic differentiation of human aortic valve interstitial cells by targeting c-KIT/interleukin-6 signaling pathway.

Zhang S, Fan L, Wang Y, Xu J, Shen Q, Xie J, Zeng Z, Zhou T
Frontiers in pharmacology 2022;13:932092

Sorafenib inhibits imatinib-resistant KIT and platelet-derived growth factor receptor beta gatekeeper mutants.

Guida T, Anaganti S, Provitera L, Gedrich R, Sullivan E, Wilhelm SM, Santoro M, Carlomagno F
Clinical cancer research : an official journal of the American Association for Cancer Research 2007 Jun 1;13(11):3363-9

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Peptide Competition and Stimulation. Extracts prepared from Mo7e cells left untreated (1) or treated with 100 ng/mL human SCF for 5 minutes (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the c-Kit [pY721] antibody (Product # 44-494G) for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to c-Kit [pY721] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of c-Kit [pY721] phosphorylation by the addition of SCF to this cell system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Peptide Competition and Stimulation. Extracts prepared from Mo7e cells left untreated (1) or treated with 100 ng/mL human SCF for 5 minutes (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the c-Kit [pY721] antibody (Product # 44-494G) for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to c-Kit [pY721] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of c-Kit [pY721] phosphorylation by the addition of SCF to this cell system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of C-Kit (pY721) was performed by loading 20 µg of A549 (lane1) and Serum Starved A549 (lane2) cell lysate using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. C-Kit (pY721) was detected at ~ 170 kDa using C-Kit (pY721) Rabbit Polyclonal Antibody (Product # 44-494G) at 1:1000 in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-c-Kit/CD117 pTyr721 Antibody was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-c-Kit/CD117 pTyr721 Antibody (Product # 44-494G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 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 Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing membranous localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of CD117 (c-Kit) [pY721] was done on A549 cells treated with EGF (200ng/mL, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with CD117 (c-Kit) [pY721] Rabbit Polyclonal Antibody (44494G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 3 c-KIT inhibition prevents the osteogenic differentiation of human valvular interstitial cells (hVICs). Western blotting (A) and immunofluorescent staining (B) were used to confirm the inhibitory effect of ISCK03 (specific inhibitor of c-KIT) on c-KIT activity. One-way ANOVA followed by a Bonferroni post hoc test . Western blotting (C) and immunofluorescent staining (D) were used to detect the protein levels of osteogenesis-specific genes (alkaline phosphatase, ALP, and runt-related transcription factor 2, RUNX2) in human valvular interstitial cells (hVICs) stimulated with osteogenic induction medium (OM) and then treated with or without ISCK03. (E) Semiquantification of the fluorescence intensity of RUNX2 and ALP. One-way ANOVA followed by Bonferroni post hoc test . (F) Alizarin red staining of mineralization nodules in hVICs stimulated with OM and then treated with or without ISCK03. One-way ANOVA followed by Bonferroni post hoc test . N = 3 per group. Values are the mean +- SD. * p < 0.05 indicates a significant difference.

44-494G

Antibody data