Protein extraction and Western blot analysis for cells were performed as previously described [53 (link)]. Frozen tumor tissues were homogenized and lysed in 5 volumes cold RIPA lysis buffer (Pierce), containing 1x phosphatase (Thermo Scientific) and protease inhibitor cocktail (Roche). Primary antibodies included: phospho-p42/p44 extracellular signal–regulated kinase (ERK) [1:1000, anti-rabbit (Cell Signaling, 4376)], total ERK [1:1000, anti-rabbit (Cell Signaling, 9102)], CDK4 [1:1000, anti-rabbit (Cell signaling, 12790)], CDK6 [1:1000, anti-rabbit (Cell signaling, 12790)], cyclin D1 [1:2000, anti-mouse (Cell Signaling, 2926)], p-S780 Rb [1:500, anti-rabbit (Abcam, ab44763)], total RB [(1:200, anti-rabbit (Santa Cruz, sc-50)], and beta actin [(1:5000, anti-mouse (Sigma, A5441). The protein-antibody complexes were detected by using an enhanced chemiluminescence kit (ThermoFisher Scientific) according to the manufacturer’s recommended protocol.
Phospho p42 p44
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-p42/p44 is a lab equipment product that detects the phosphorylation of p42 and p44 mitogen-activated protein kinases (MAPK). It is used to monitor the activation state of these proteins, which are important signaling molecules involved in cellular processes such as proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Phospho p38, by Cell Signaling Technology (3 mentions)
Phospho jnk1 2, by Cell Signaling Technology (2 mentions)
Irdye 680 or 800 secondary antibodies, by Rockland Immunochemicals (2 mentions)
On targetplus ask1 sirna, by Horizon Discovery (2 mentions)
Recombinant tgf β1, by R&D Systems (2 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (2 mentions)
Tc ask 10, by Bio-Techne (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
P s780 rb, by Abcam (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Erk p44 p42, by Cell Signaling Technology (1 mentions)
Sb203580, by Merck Group (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Supersignal west pico reagent, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Merck Group (1 mentions)
Anti inos, by Novus Biologicals (1 mentions)
Cd206, by Abcam (1 mentions)
6 protocols using phospho p42 p44
1
Western Blot Analysis of Tumor Tissues
2
Mapping MAPK Signaling Pathways in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of MAPK activation, whole cell lysate was subjected to 12% SDS-PAGE and transferred to a PVDF membrane. Rabbit polyclonal antibodies as the primary antibodies against signaling molecules including ERK (p42/p44), phospho-p42/p44, p38, phospho-p38, JNK, and phospho-JNK, were purchased from Cell Signaling Technology (Danvers, MA). Mouse polyclonal antibodies against GAPDH were purchased from Sigma-Aldrich. After incubation with the appropriate primary and horseradish peroxidase-conjugated secondary antibodies, blots were developed using SuperSignal West Pico reagents (Thermo Scientific, Logan, Utah, USA).
To identify the critical MAPK signaling pathway activated by rSSB, cells were preincubated with SB203580 (specific p38 inhibitor, Sigma-Aldrich), PD98059 (specific MEK-1 inhibitor, Sigma-Aldrich), or pertusis toxin (specific Gαi-protein-coupled receptor inhibitor, Calbiochem, San Diego, CA, USA) for 1 hour, and then incubated with rSSB or LPS for 2 hours.
To identify the critical MAPK signaling pathway activated by rSSB, cells were preincubated with SB203580 (specific p38 inhibitor, Sigma-Aldrich), PD98059 (specific MEK-1 inhibitor, Sigma-Aldrich), or pertusis toxin (specific Gαi-protein-coupled receptor inhibitor, Calbiochem, San Diego, CA, USA) for 1 hour, and then incubated with rSSB or LPS for 2 hours.
3
Immunohistochemical Analysis of Lung Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human lung samples from donors and COPD patients were used. The study complied with the Declaration of Helsinki, and the tissue donation protocol was approved by the ethics committee of the faculty of medicine at Justus-Liebig University of Giessen, Germany. Immunohistochemical staining was performed as reported previously [25 (link)] with subtle modifications. Primary antibodies were used as follows: CD68 (Cat#ab955, Abcam, Cambridge, UK; 1:200); CD206 (Cat#ab64693, Abcam, Cambridge, UK; 1:100), anti-iNOS (Cat#NB300–605, Novus Biologicals, Littleton, CO, USA; 1:250), phospho p42/p44 (Cat#4370, Cell Signaling Technology, Danvers, MA, USA; 1:200).
4
ASK1 Regulation of Cell Signaling
5
ASK1 Regulation of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against rabbit total and phospho-apoptosis signal-regulating kinase 1 (ASK1), phospho-p42/p44, phospho-p38, phospho-JNK1/2 antibodies were from Cell Signaling Technology (Beverly, MA, USA). IRDye 680 or 800 secondary antibodies were from Rockland (Gilbertsville, PA, USA). CyQUANT cell proliferation assay kit was purchased from Life Technologies (Grand Island, NY, USA). TC ASK 10, an ASK1 inhibitor was purchased from Tocris Biosciences, USA, recombinant TGFβ1 was obtained from R&D Systems, Minneapolis, MN, USA. ON-TARGETplus ASK1 siRNA was purchased from Dharmacon, Mulgrave, Victoria, Australia. Lipofectamine RNAiMAX was purchased from Invitrogen, Australia. All other chemicals were of analytical grade and were obtained from Sigma Aldrich, USA.
6
Ganetespib-Induced Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated into 10 cm dishes and grown to sub confluence. Ganetespib (1–100 nM) was added to the medium 24 hours after plating. Cells were harvested, homogenized 24 hours later and 50 µg of total protein was loaded into each well of an 8–12% polyacrylamide gel and separated. Protein was transferred onto a polyvinylidene fluoride (BioRad) blotting membrane and blocked for an hour using 5% BSA or milk in TBST (tris-buffered saline supplemented with 0.1% Tween-20). Phospho-antibodies were incubated with 5% BSA in TBST, other antibodies were incubated with 5% milk in TBST. The membranes were probed with antibodies for phospho-S6 (Cell Signaling), ATM (Millipore), Wee1 (Cell Signaling), caspase 3 (Cell Signaling), phospho-Chk1-Ser345 (Santa Cruz), Chk1 (Santa Cruz), Hsp72 (Selleck chemicals), p42/44 (Cell Signaling), phospho-p42/p44 (Cell Signaling), and subsequently with horseradish peroxidase-labeled mouse anti-rabbit secondary antibodies (Sigma-Aldrich). Each antibody incubation step was followed by 3–4 washes with TBST. The secondary antibody was then coupled with GE ECL Plus kit (GE Life Sciences) and protein levels were detected using autoradiography films (Denville Scientific, Inc.). Experiments were done at least twice.
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!