Anti phospho kit

Manufactured by Cell Signaling Technology

Sourced in Switzerland, United States

Anti-phospho-KIT is a primary antibody that specifically recognizes the phosphorylated form of the KIT protein. KIT is a receptor tyrosine kinase that plays a critical role in various cellular processes. This antibody can be used to detect and analyze the phosphorylation status of KIT in various experimental applications.

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Close spelling variants of this product

Anti-phospho-EGFR sampler kit Anti-phospho-c-Kit

4 protocols using anti phospho kit

Investigating Mutant Kinase Signaling

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Exponentially growing Molm14, HB119, or Ba/F3 cells stably expressing mutant isoforms were plated in RPMI medium 1640 + 10% (vol/vol) FCS supplemented with PKC412 or Star 27 at the indicated concentration. HMC1.2 cells were cultured and treated in IMDM + 10% FCS. After a 90-min incubation, the cells were washed in PBS, lysed, and processed as previously described. Immunoblotting was performed using anti-phospho-FLT3, anti-phospho-KIT, anti-phospho-STAT5, anti-STAT5, anti-phospho-ERK, anti-ERK, anti-phospho-S6, anti-S6, anti-KIT (Cell Signaling, Beverley, MA) and anti-FLT3 S18 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Warkentin A.A., Lopez M.S., Lasater E.A., Lin K., He B.L., Leung A.Y., Smith C.C., Shah N.P, & Shokat K.M. (2014). Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy. eLife, 3, e03445.

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Analyzing SCF and STI571 Effects

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After serum starvation for 12 h, cells were incubated for 4 h with 10 ng/mL SCF
and/or 250 nM STI571 (Novartis Pharmaceuticals, Basel, Switzerland), and
immunoblot analysis was conducted as described previously51 (link).
Primary antibodies including anti-phospho-KIT, anti-total/phospho-Akt,
anti-total/phospho-S6 ribosomal protein, and β-actin antibodies
purchased from Cell Signaling Technologies (Beverly, MA), and an anti-KIT
antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Amagai Y., Matsuda A., Jung K., Oida K., Jang H., Ishizaka S., Matsuda H, & Tanaka A. (2015). A point mutation in the extracellular domain of KIT promotes tumorigenesis of mast cells via ligand-independent auto-dimerization. Scientific Reports, 5, 9775.

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Western Blot Analysis of KIT Signaling

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Cells were washed in PBS and lysed in Triton-x Lysis Buffer supplemented with protease and phosphatase inhibitors. Lysate concentrations were measured using the Pierce BCA™ Protein Assay. Protein was subjected to electrophoresis on a 4–12% Bis-Tris Gel with MES buffer according to manufacturer guidelines using the Invitrogen Bolt. Following electrophoresis, proteins were transferred to a nitrocellulose membrane on the Invitrogen iBlot and underwent blocking using iBind solution. Antibody probing was performed using iBind with anti-KIT (Cell Signaling), anti-Phospho-KIT (Cell Signaling), anti-β-actin AC-15 (Sigma-Aldrich,), HRP Goat anti-Mouse IgG (H+L) secondary (ThermoFisher), and HRP Goat anti-Rabbit IgG (H+L) secondary (ThermoFisher). Blots were developed in SuperSignal West Pico Chemiluminescent Substratea and imaged with Bio-Rad ChemiDoc™ MP.

Tarlock K., Alonzo T.A., Wang Y.C., Gerbing R.B., Ries R., Loken M.R., Pardo L., Hylkema T., Joaquin J., Sarukkai L., Raimondi S.C., Hirsch B., Sung L., Aplenc R., Bernstein I., Gamis A.S., Meshinchi S, & Pollard J.A. (2019). Functional Properties of KIT Mutations are Associated with Differential Clinical Outcomes and Response to Targeted Therapeutics in CBF Acute Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(16), 5038-5048.

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Western Blot Analysis of Signaling Proteins

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Western Blot analysis was performed according to standard procedures on lysates from HMC-1.1 cells and from excised tumors (13) . Membranes were probed with the following antibodies: antiphospho-SHP-1 and anti-SHP-1 (each 1:200, Santa Cruz Technology, Santa Cruz, CA, USA), antiphospho-Bcl2 (Ser 70), Bcl2, anti-phospho-KIT and anti-KIT (each 1:1000, Cell Signaling Technology), anti-Vinculin and anti-GAPDH (1:1000, Santa Cruz Technology). Signal was acquired using BioRad Chemi Doc XRS + Gel imaging system and bands quantified using Image Lab software.

Landolina N., Zaffran I., Smiljkovic D., Serrano-Candelas E., Schmiedel D., Friedman S., Arock M., Hartmann K., Pikarsky E., Mandelboim O., Martin M., Valent P, & Levi-Schaffer F. (2020). Activation of Siglec-7 results in inhibition of in vitro and in vivo growth of human mast cell leukemia cells. Pharmacological research, 158.

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