ALK protein expression was investigated by IHC methods both in cell lines and tumor samples. Briefly, three to four micron-thick FFPE sections were unmasked with EDTA buffer at pH8 for 30 minutes, made react with the ALK antibody (clone 5A4, Santa Cruz, Heidelberg, Germany, dilution 1:100 for 60 min) and then incubated with a commercially available detection kit (EnVision™ FLEX+, Dako, Glostrup, Denmark) in an automated immunostainer (Dako Autostainer System). IHC results were scored as positive (more than 10% reactive neoplastic cells) or negative (10% or less reactive neoplastic cells). Positive results were further defined on the basis of the distribution of reactivity (focal or diffuse), intensity (weak, moderate, strong), and cellular site (cell membrane, cytoplasm, nucleus). Positive and negative controls were used as appropriate. Moreover, Phospho-ALK expression by IHC was studied in the ALK rearranged case utilizing the antibody Phospho-ALK (tyr 1604, Cell Signaling, dilution 1:5 for 120 min) with a few modification of the protocol (unmasked with Dako PT-link, EnVision™ FLEX Target Retrieval Solution, High pH – 60 mi at 96°C).
Phospho alk
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-ALK is a lab equipment product that detects phosphorylated forms of the anaplastic lymphoma kinase (ALK) protein. It is used to analyze the activation status of the ALK protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (4 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Phospho ros1, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pt link, by Agilent Technologies (1 mentions)
Envision flex target retrieval solution, by Agilent Technologies (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Autostainer system, by Agilent Technologies (1 mentions)
Phospho erk5, by Cell Signaling Technology (1 mentions)
Pan erk, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Phospho igf 1r, by Cell Signaling Technology (1 mentions)
Proteome profiler human phospho kinase array kit, by R&D Systems (1 mentions)
Phospho egfr, by Cell Signaling Technology (1 mentions)
Phospho her3, by Cell Signaling Technology (1 mentions)
Total egfr, by Cell Signaling Technology (1 mentions)
Igf 1r, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence plus reagent, by GE Healthcare (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
7 protocols using phospho alk
1
Immunohistochemical Analysis of ALK Expression
2
Neuroblastoma Cell Lines and Antibody Analysis
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Neuroblastoma cell lines used were CLB-BAR (amplified MYCN/ALK, Δ4-11), CLB-GE (amplified MYCN/ALK, ALK-F1174V), CLB-PE (amplified MYCN, WT ALK) and IMR-32 (amplified MYCN, WT ALK) and PC12 cells were cultured and grown as previously reported [7 (link), 35 (link)]. Primary antibodies employed for immunoblotting were: phospho-ALK (Y1604), phospho-AKT (S473), MYCN, phospho-ERK1/2 (1:2000), phospho-ERK5 (1:1000), ERK5, panERK, Actin (1:5000) from Cell Signaling Technology (Danvers, MA). Monoclonal antibody 135 (anti-ALK) was produced in the Hallberg laboratory against the extracellular domain of ALK [38 (link)]. Horseradish peroxidase conjugated secondary antibodies; goat anti-rabbit IgG and goat anti-mouse, IgG (1:5000) were obtained from Thermo Scientific. Brigatinib, was obtained from Ariad Pharmaceuticals and crizotinib was from Haoyuan Chemexpress Co., Limited, Shanghai.
3
Kinase Phosphorylation Profiling Assay
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Total protein was isolated from cells as indicated in each experiment. The following primary antibodies were used and purchased from Cell Signaling Technology (Beverly, MA): rabbit anti-total EGFR, total ALK, total HER3, total IGF-1R, total ERK1/2, total AKT, phospho-ALK (pTry1604), phospho-EGFR (pTyr1068), phospho-HER3 (pTyr1222), phospho-IGF-1R (pTry1135), phospho-ERK1/2 (pThr202/Tyr204), and phospho-AKT (pSer473). The primary antibodies were diluted 1:1000 in blocking buffer and incubated with the membranes overnight at 4°C. Membranes were washed with Tris-Buffered Saline and Tween 20 (TBST) buffer three times, and goat anti-rabbit IgG secondary antibody was applied for 1 hour at room temperature. HRP-conjugated anti-GAPDH antibody was purchased from Life Technologies and diluted 1:2000 for use.
To detect phosphorylation of human kinases, the Proteome Profiler Human Phospho-Kinase Array Kit from R&D Systems (Minneapolis, MN) was employed. For this assay, 300 μg of total proteins was isolated from cells, and the Phospho-Kinase Array Kit was used according to manufacturer’s instructions to detect the phosphorylation of 43 human kinases.
To detect phosphorylation of human kinases, the Proteome Profiler Human Phospho-Kinase Array Kit from R&D Systems (Minneapolis, MN) was employed. For this assay, 300 μg of total proteins was isolated from cells, and the Phospho-Kinase Array Kit was used according to manufacturer’s instructions to detect the phosphorylation of 43 human kinases.
4
Immunoblotting and Phospho-Receptor Tyrosine Kinase Profiling
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The following antibodies were obtained from Cell Signaling Technology: phospho‐EGFR (Tyr1068), EGFR, phospho‐ALK (Tyr1282/1283), ALK, phospho‐ROS1 (Tyr2274), ROS1, phospho‐ERK1/2 (Thr202/Tyr204), ERK1/2, phospho‐AKT (Ser473), AKT, GAPDH, and horseradish peroxidase‐conjugated anti‐rabbit IgG antibody.
For immunoblotting, cells were harvested, washed in phosphate‐buffered saline, and lysed in radioimmunoprecipitation assay buffer (1% Triton X‐100, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L β‐glycerol‐phosphate, 10 mmol/L NaF, and 1 mmol/L sodium orthovanadate) containing a protease inhibitor cocktail (Roche Applied Sciences). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto membranes, which were then incubated with the indicated primary and secondary antibodies. Chemiluminescence was detected using an enhanced chemiluminescence plus reagent (GE Healthcare Biosciences).
Phospho‐receptor tyrosine kinase arrays were performed using a phospho‐receptor tyrosine kinase array kit (R&D Systems) in accordance with the manufacturer's instructions. Bands and dots were detected using the ImageQuant LAS‐4000 imager (GE Healthcare Biosciences).
For immunoblotting, cells were harvested, washed in phosphate‐buffered saline, and lysed in radioimmunoprecipitation assay buffer (1% Triton X‐100, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L β‐glycerol‐phosphate, 10 mmol/L NaF, and 1 mmol/L sodium orthovanadate) containing a protease inhibitor cocktail (Roche Applied Sciences). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto membranes, which were then incubated with the indicated primary and secondary antibodies. Chemiluminescence was detected using an enhanced chemiluminescence plus reagent (GE Healthcare Biosciences).
Phospho‐receptor tyrosine kinase arrays were performed using a phospho‐receptor tyrosine kinase array kit (R&D Systems) in accordance with the manufacturer's instructions. Bands and dots were detected using the ImageQuant LAS‐4000 imager (GE Healthcare Biosciences).
5
Antibody Panel for Cellular Signaling
6
ALK Phosphorylation in Ba/F3 Cells
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Ba/F3 cells (1 × 106) were seeded into 12‐well plates and treated with different drugs at different concentrations for three hours. Cells were suspended in lysis buffer containing 0.1 M Tris (pH 7.5), 10% glycerol and 1% sodium dodecyl sulfate (SDS), and boiled at 100°C for five minutes. The protein concentrations were measured with a BCA protein assay Kit (Thermo Fischer Scientific). The lysates were adjusted to 0.9 μg/μL with lysis buffer and 20% volume of the sample buffer containing 0.65 M Tris (pH 6.8), 20% 2‐mercaptoethanol, 10% glycerol, 3% SDS, and 0.01% bromophenol blue was added. Next, 10 μg of each sample was electrophoresed using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotted with the antibodies against total‐ALK (#3633S Cell Signaling Technology), phospho‐ALK (#3341 Cell Signaling Technology), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (MAB374; Millipore, Billerica, MA, USA).
7
Western Blot Analysis of Signaling Proteins
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Protein extracts were prepared by washing twice in cold PBS followed by lysis with SDS-lysis buffer (50 mM Tris-HCl, pH7.4, 2%SDS). Cell lysates were boiled for 10 min and cleared by centrifugation at 14,000× g for 5 min at 4°C. The supernatant was collected and subsequently resolved by SDS-PAGE and transferred to nitrocellulose membranes, probed with the appropriate primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunoreactive proteins were detected using an ECL plus detection reagent (Picece, Rockford, IL) and imaged by autoradiography.
Antibodies against GAPDH, Cdc2, Cyclin B1, Hsp90α and Hsp70 were from Epitomics (Burlingame, CA). Antibodies against c-Met, phospho-Met (Y1234/1235), EGFR, phospho-EGFR (Y1068), HER2, phospho-HER2 (Y1221/1222), ALK, phospho-ALK (Y1604), phospho-Cdc2 (Y15), Cdc25c, phospho-Cdc25c (S216), AKT, phospho-AKT (S473), ERK, phospho-ERK (T202/Y204), Cleaved caspase-3, Caspase-8, Caspase-9, PARP, Bcl-2, Bax and β-actin were from Cell Signaling Technology (Beverly, MA).
Antibodies against GAPDH, Cdc2, Cyclin B1, Hsp90α and Hsp70 were from Epitomics (Burlingame, CA). Antibodies against c-Met, phospho-Met (Y1234/1235), EGFR, phospho-EGFR (Y1068), HER2, phospho-HER2 (Y1221/1222), ALK, phospho-ALK (Y1604), phospho-Cdc2 (Y15), Cdc25c, phospho-Cdc25c (S216), AKT, phospho-AKT (S473), ERK, phospho-ERK (T202/Y204), Cleaved caspase-3, Caspase-8, Caspase-9, PARP, Bcl-2, Bax and β-actin were from Cell Signaling Technology (Beverly, MA).
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