The following antibodies for histology and cell staining were purchased from Santa Cruz Biotechnology: anti-MUC5AC (Santa Cruz, CA, sc-16903, AB_649616), anti-phospho-PI3K p85α (Tyr467: Santa Cruz, CA, sc-293115, AB_10844180), anti-PI3K p85α (Santa Cruz, CA, sc-31970, AB_2268186), anti-phospho-Akt1 (Thr308: Santa Cruz, CA, sc-135650, AB_2224730), anti-Akt1 (Santa Cruz, CA, sc-1618, AB_630849), anti-phospho-NFκB p65 (Ser536: Santa Cruz, CA, sc-33020, AB_2179018), anti-NFκB p65 (Santa Cruz, CA, sc-109, AB_632039), anti-Lyn (Santa Cruz, CA, sc-15, AB_2281450), and anti-β-actin (Santa Cruz, CA, sc-130656, AB_2223228). The following antibodies for histology were purchased from Abcam Biotechnology: anti-BIP (Abcam, ab21685, AB_2119834), anti-CHOP (Abcam, ab11419, AB_298023), anti-histone H3 (Abcam, ab1791, AB_302613), and anti-IL-13 (Abcam, ab133353, AB_11157609). Anti-phospho-Lyn (Tyr416: Cell Signaling Technology, #2101, AB_331697) was purchased from Cell Signaling Technology. Anti-IL13 (R&D Systems, AF-413-NA, and AB_2124173) and IL-13 ELISA reagents (R&D Systems, M1300CB) were purchased from R&D Systems. IL-13 (PeproTech, #200-13), 4-Phenylbutyric acid (4-PBA, Sigma-Aldrich, P21005), PI3K Inhibitor PI-103 (Selleck, S1038) were purchased as indicated. A nonsilencing siRNA control and a Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti phospho lyn
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Lyn is a primary antibody that detects Lyn protein when phosphorylated. Lyn is a Src family kinase that plays a role in signal transduction pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho syk, by Cell Signaling Technology (3 mentions)
Anti phospho erk, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti phospho cpla2, by Cell Signaling Technology (2 mentions)
Anti phospho fyn, by Cell Signaling Technology (2 mentions)
Anti phospho plcγ1, by Cell Signaling Technology (2 mentions)
Anti cox 2, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Anti lyn, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti lyn, by Santa Cruz Biotechnology (1 mentions)
Anti bip, by Abcam (1 mentions)
Anti muc5ac, by Santa Cruz Biotechnology (1 mentions)
Anti pi3k p85α, by Santa Cruz Biotechnology (1 mentions)
Anti phospho nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti chop, by Abcam (1 mentions)
Anti il 13, by Abcam (1 mentions)
Anti il 13, by R&D Systems (1 mentions)
Af 413 na, by R&D Systems (1 mentions)
6 protocols using anti phospho lyn
1
Antibody-Based Histology and Cell Staining Protocols
2
Immunoblot Analysis of Signaling Pathways
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The cells were scraped from the plates using RIPA Lysis Buffer (Merck Millipore, Darmstadt, Germany) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After incubation on ice for 30 min, the cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were quantified using BCA protein assay (Thermo Scientific, Waltham, MA, USA). The protein lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Anti-COX-2, anti-phospho-cPLA2, anti-phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-ERK, anti-phospho-Akt, and anti-α-tubulin antibodies (Cell Signaling Technology, Boston, MA, USA) were used to detect COX-2, p-cPLA2, p-Lyn, p-Syk, p-Fyn, p-PLCγ1, p-ERK, p-Akt, and α-tubulin, respectively. α-Tubulin was used as protein loading control. Blots were observed using a western blot detection kit (Thermo Scientific, Waltham, MA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories, Richmond, CA, USA).
3
Investigating Phosphorylation Signaling Pathways
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The antibodies used were anti-phospho-Stat3 (S727), anti-Stat3, anti-phospho-PI3K, anti-PI3K, anti-phospho-Erk (Y204), anti-Erk, anti-phospho-Lyn (Y507), anti-Lyn, anti-phospho-Akt (S473), anti-Akt (all from Cell Signaling Technology) and anti-Actin (BD Biosciences). Gel images were analyzed with ImageJ (NIH) for quantification.
4
Immunoblot Analysis of Signaling Proteins
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The cells were scraped from the plates with RIPA lysis buffer containing a protease and phosphatase-inhibitor cocktail (Roche, Basel, Switzerland). The lysates were quantified using the BCA protein assay kits (Thermo, MA, USA). Equal amounts of protein were resolved on 10% SDS-PAGE and then transferred to a PVDF membrane. Anti-COX-2, anti-β-actin, anti- phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-cPLA2, anti-phospho-ERK, and anti-phospho-Akt (Cell Signaling, MA, USA) were used to detect COX-2, β-actin and the phosphorylated form of Lyn, Syk, Fyn, PLCγ1, cPLA2, ERK, and Akt, respectively.
5
Immunoblotting Analysis of Inflammatory Signaling
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DNCB, anti-DNP-IgE and DNP-HSA were purchased from Sigma-Aldrich (St. Louis, MO, USA), Eucalyptus oil, procured from Nippon Terpene Chemicals (Tokyo, Japan), conforms with the Japanese Pharmacopoeia, and its main component, 1,8-cineole, accounts for 82.5% of the oil. 1,8-cineole was obtained from Nacalai Tesque (Kyoto, Japan), and dimethyl sulfoxide (DMSO) was acquired from Wako (Osaka, Japan). The following antibodies for immunoblotting were purchased from Cell Signalling Technology (Beverly, MA, USA): anti-Syk, anti-phospho-Syk, anti-Lyn, anti-phospho-Lyn, anti-PLA2, anti-phospho-PLA2, anti-PLCγ, anti-phospho-PLCγ, anti-p38 and anti-phospho-p38. Goat anti-rabbit IgG horseradish peroxidase was purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Surface and Intracellular Phosphatase Analysis
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For surface expression analysis, cells were prepared as outlined above and 1 × 106 were aliquoted into FACS tubes. Cells were pelleted by centrifugation and each tube was resuspended in flow buffer [1% (w/v) BSA, 4 mmol/L EDTA, and 0.15 mmol/L NaN3 in PBS] containing anti-CD19 and anti-CD5 antibodies for CLL cell detection. Antibodies against specific key markers and surface phosphatases were then used in various combinations. Cells were incubated on ice in the dark for 15 minutes prior to washing with flow buffer. Cells were resuspended in flow buffer and analyzed using a Canto cytometer. Data were analyzed using FlowJo.
For intracellular phosphatases, lysates were analyzed by immunoblotting with the following antibodies: anti-phospho-SHIP1, anti-phospho-SHP1, anti-phospho-LYN (Y507; all Cell Signaling Technology), anti-SHP1, anti-phospho-LYN (Y396; both Abcam), anti-SHIP1, anti-LYN (both Insight Biotechnology), anti-phospho-PTPN22 (R&D Systems), and anti-GAPDH (Thermo Fisher Scientific). Primary antibodies were probed using species-specific HRP-conjugated secondary antibodies (Dako) and viewed using a Chemidoc imager (Bio-Rad).
For intracellular phosphatases, lysates were analyzed by immunoblotting with the following antibodies: anti-phospho-SHIP1, anti-phospho-SHP1, anti-phospho-LYN (Y507; all Cell Signaling Technology), anti-SHP1, anti-phospho-LYN (Y396; both Abcam), anti-SHIP1, anti-LYN (both Insight Biotechnology), anti-phospho-PTPN22 (R&D Systems), and anti-GAPDH (Thermo Fisher Scientific). Primary antibodies were probed using species-specific HRP-conjugated secondary antibodies (Dako) and viewed using a Chemidoc imager (Bio-Rad).
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