Antibodies directed against IκBα, phospho-ERK1/2 (Thr202/Tyr204), phospho-IκBα (Ser32), phospho-IKKα/β (Ser176/180), phospho-LAT1 (Tyr191), phospho-p38 (Thr180/Tyr182), phospho-PKB (Ser473), phospho-tyrosine (P-Tyr-100) were from Cell Signaling Technology (Frankfurt, Germany). Antibodies against phospho-PLC-γ1 (Tyr783; sc-12943-R), GAPDH (6C5) and SHIP1 (P1C1) were obtained from Santa Cruz (Heidelberg, Germany) and antibodies against PI3K p85 (06-195) from EMD Millipore (Merck, Darmstadt, Germany). DNP-HSA containing 30-40 moles DNP per mole albumin and monoclonal IgE with specificity for DNP (SPE-7) were purchased from SIGMA (Deisenhofen, Germany). SPE-7 is a potent cytokinergic IgE and free SPE-7 was shown to cross-link FcεRI-bound SPE-7 by Fv-Fv interactions [46 (link)]. For this cytokinergic action the used concentration of SPE-7 (>1.0 μg/ml) had to be high enough to saturate all FcεRIs [46 (link)]. Therefore, to prevent interference with such cytokinergic action, we used only 0.15 μg/ml SPE-7 for preloading overnight for all experiments. 0.15 μg/ml SPE-7 was shown to be not sufficient to saturate all FcεRIs on BMMCs (data not shown). DMSO was obtained from Carl Roth GmbH & Co (Karlsruhe, Germany), Wortmannin from Calbiochem (Schwalbach, Germany), Cyclosporine A from Enzo Lifesciences (Lörrach, Germany), and INCA-6 from Tocris Bioscience (Wiesbaden-Nordenstadt, Germany).
Ship1 p1c1
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
SHIP1 P1C1 is a laboratory equipment product offered by Santa Cruz Biotechnology. It serves as a tool for researchers to study the function and regulation of the SHIP1 (Src homology 2 domain-containing inositol phosphatase 1) protein.
Automatically generated - may contain errors
Lab products found in correlation
Dnp hsa, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
Cyclosporine a, by Enzo Life Sciences (1 mentions)
Inca 6, by Bio-Techne (1 mentions)
Thunder imaging system, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Stellaris 5 confocal microscope, by Leica (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Femto ecl, by Thermo Fisher Scientific (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Non fat milk, by Santa Cruz Biotechnology (1 mentions)
Hsp90α β f 8, by Santa Cruz Biotechnology (1 mentions)
Nonfat milk, by Cell Signaling Technology (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
M iggk bp hrp, by Santa Cruz Biotechnology (1 mentions)
3 protocols using ship1 p1c1
1
Signaling Pathway Activation in Mast Cells
2
Immunofluorescence Staining of SHIP1 and PI(3,4)P2
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Cells were fixed with 4% PFA for 20 min at RT. After 15 min of permeabilization (PBS, Triton 0.25%), samples were incubated with Blocking Buffer (BF = PBS, Triton 0.25%, BSA 2%) for 1h at RT. Cells were incubated with primary antibody ON at 4 °C (SHIP1 P1C1, 1:200 in BF, Santa Cruz Biotechnology; PI(3,4)P2 Purified Anti-PtdIns(3,4)P2 IgG, Echelons Biosciences, #Cat Z-P034, final concentration 5 μg/mL). After three washes with PBS, Alexa Fluor Plus 555 Goat anti-Mouse IgG (H + L) (1:1000 in BF, Thermofisher Scientific) was added on the samples for 1h at RT. Other three washes with PBS were performed, followed by DAPI incubation (1 μg/ml in PBS, Thermo Fisher Scientific) for 10 min at RT. Cells were rinsed once with PBS and coverslips were mounted on glass slide with Mowiol. Images were acquired with a fluorescence microscope (Thunder Imaging System, Leica Microsystems) or with a Leica Stellaris 5 Confocal microscope (Leica Microsystems), with APO 63x/1.40 oil objective. Two biological replicates per condition were acquired and analyzed. Image analysis was performed using ImageJ software. Fixed thresholds for signal intensity were applied across experiments, and area covered was measured per field of view, then rationalized for the number of cells, given by DAPI counting.
3
Western Blot Analysis of SHIP1 Protein
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Cells were harvested from an 80% confluent P2 culture. After two washes with cold PBS, cells were spun down at 10,000xg for 1 min and lysed on ice for 20 min with Lysis buffer (20mM Tris, 150mM NaCl, 1mM EGDA, 1mM EGTA, 1%Triton X-100) containing 1x protease and phosphatases inhibitor Halt (ThermoFisher Scientific). The cell lysate was spun down for 20 min at 10,000xg to eliminate cell debris. Western blot was then performed as described previously (Pedicone et al., 2019 (link)). Briefly, 25μg of cleared lysate in loading buffer containing DTT was loaded on 4-15% Precast gels (Bio-Rad). The gel was run 30 min at 180V followed by transfer to a nitrocellulose membrane with Trans-Blot Turbo (Bio-Rad). The membrane was blocked in 5% Nonfat milk (Cell Signaling) in TBST (Tris-Buffer Saline, 0.1% Tween 20) O/N and incubated 2h with 1:500 SHIP1 P1C1 (Santa Cruz Biotechnology), 1:1000 β-actin C-4 (Santa Cruz Biotechnology), or 1:1000 HSP90 α−β F-8 (Santa Cruz Biotechnology). After three-10-min washes in TBST the membrane was incubated with1:1000 m-IgGkBP-HRP (Santa Cruz Biotechnology) in 5% Nonfat milk for 45 min. To detect the chemiluminescence signal, we used a Bio-Rad Chemidoc with ECL Pico(ThermoFisher Scientific) for housekeeping proteins and ECL Femto (ThermoFisher Scientific) for SHIP1.
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