44-500G

antibody from Invitrogen Antibodies

Targeting: KIT C-Kit, CD117, PBT, SCFR

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

44-500G

Provider

Invitrogen Antibodies

Product name

Phospho-c-Kit (Tyr936) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

References

Submitted references

CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

Moiseeva EP, Straatman KR, Leyland ML, Bradding P
PloS one 2014;9(1):e85980

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition: Extracts prepared from M07e cells left untreated (1) or treated (2-5) with SCF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of K562 (Lane1), K562 treated for 10 minutes with 100 ng/mL of SCF (Lane 2), HT 29 (Lane 3) and HT 29 treated for 10 minutes with 100 ng/mL of SCF (lane 4). The blots were probed with Anti-c-Kit (pY936) Rabbit Polyclonal Antibody (Product # 44-500G, 1:250 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 150 kDa band corresponding to c-Kit (pY936) was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by wet transfer (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Phospho-c-Kit/CD117 pTyr936 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 pTyr936 Antibody (Product # 44-500G) 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 cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of CD117 (c-Kit) [pY936] 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) [pY936] Rabbit Polyclonal Antibody (44500G, 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.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2 Modulated CADM1 levels in HMC-1 cells influenced surface Kit expression, an assembly of filamentous actin and tyrosine phosphorylation. A. HMC-1 cells were transduced with SP4, SP1, control shRNA LucSh, CADM1 shRNA (Sh5 or Shm) viral particles. Then examined for expression of surface CADM1 (total n = 30 groups from 7 transductions), surface Kit (n = 27 from 7 transductions) and amounts of F-actin (n = 23 from 4 transductions) by FACS. The control group combines LucSh-transduced and non-transduced cells, CADM1 downregulated group combines Sh5- and Shm-transduced cells. All data were expressed as a percentage of the levels in the SP-overexpressing cell group. B and C. Scatter plots for the data presented in A with correlation or regression model parameters are shown for Kit ( B ) and F-actin ( C ) as a function of CADM1. Data for SP4- and SP1-expressing cells are shown in different colours. D. Western blotting of protein extracts from LucSh-, SP4- and Shm-transduced HMC-1 cells, with 3 independent transductions for each group, developed with Abs shown on the right of the blots. E. Protein bands, shown in D and Fig. S 2C , were quantified. Bands in SP4 (n = 5) and Shm (n = 4) groups were expressed as percentages of control LucSh/GFP group (n = 5) for each protein, except phosphotyrosine 58-63 kDa bands (n = 3). * P