Where indicated, cells were treated with tyrosine kinase inhibitor for 2 to 4 hours. Lysates were prepared from cells using a standard cell lysis buffer as described before (21 (link)). Protein quantitation of cleared cell lysates was performed using Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific), and 25 μg of total protein was loaded on pre-cast 4–12% Criterion™ XT Bis-Tris Protein Gels (Bio-Rad, # 3450125). Proteins were transferred to nitrocellulose membranes, and probed with phospho-ROS1 [#3078, 1:1,000; Cell Signaling Technology (CST)], total ROS1 (#3266, 1:1,000; CST), phospho-SHP2 (#3751, 1:1000, CST), total SHP2 (#3397, 1:1000; CST), phospho-ERK1/2 (#9101, 1:1,000; CST), total ERK2 (sc-1647, 1:2,000; Santa Cruz, phospho-Akt (#4060, 1:1,000; CST), AKT (#610860, 1:1,000; BD Transduction Laboratories). Blots were imaged using either a LI-COR Odyssey imaging system or the Bio-Rad ChemiDoc imaging station according to the manufacturer’s protocol for immunoblot detection with use of infrared dye or horseradish peroxidase-conjugated secondary antibodies, respectively. phospho-ROS1 detection required the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific).
Phospho ros1
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-ROS1 is a primary antibody that recognizes the phosphorylated form of the ROS1 protein. ROS1 is a receptor tyrosine kinase that is involved in various cellular processes. The Phospho-ROS1 antibody can be used to detect and quantify the phosphorylated state of the ROS1 protein using techniques such as Western blotting or immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging station, by Bio-Rad (2 mentions)
Total erk2, by Santa Cruz Biotechnology (2 mentions)
Odyssey imaging system, by Bio-Rad (2 mentions)
Phospho alk, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Criterion xt bis tris protein gel, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
P akt1 2 3 antibody ser 473 r sc 7985 r, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse immunoglobulin ig g, by ICN Biomedicals (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by ICN Biomedicals (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Santa Cruz Biotechnology (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence plus reagent, by GE Healthcare (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Phospho receptor tyrosine kinase array kit, by R&D Systems (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ptp1b, by Abcam (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
5 protocols using phospho ros1
1
Tyrosine Kinase Inhibitor Effects on Signaling
2
Western Blotting Methodology in ROS1 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described previously.24 (link)–26 (link) Monoclonal antibody (mAb) against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (G-9) (sc-365062) was used to standardize cytosolic protein loading on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ROS1 (D4D6) rabbit mAbs from Cell Signaling Technology (catalog no: 3287) were used to recog-nize FIG-ROS1 and Phospho-ROS1 activity (Tyr2274) (catalog no: 3078). Anti-GOPC/FIG antibody (MyBioSource: MBS421395) was used to detect FIG expression. p-Met antibody (Tyr 1365) (sc-3408), p-Met (F-5) (sc-377548), p-ALK antibody (Tyr 1586) (sc-109905), Bim (H-191) (sc-11425), survivin (D-8) (sc-17779), Mcl-1 antibody (S-19) (sc-819), and p-Akt1/2/3 antibody (Ser 473)-R (sc-7985-R) were purchased from Santa Cruz Biotechnology, Inc. The secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig) G (ICN Biomedicals) and horseradish peroxidase-conjugated goat anti-rabbit IgG (ICN Biomedicals).
3
Inhibitor Effects on CD74-ROS1 Signaling
4
Immunoblotting and Phospho-Receptor Tyrosine Kinase Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!