SET antagonist TD19 (EMQA), a derivative of erlotinib with chemical name of N4-(3-ethynylphenyl)-6,7-dimethoxy-N2-(4-phenoxyphenyl) quinazoline-2,4-diamine), was kindly provided from Dr. Chung-Wai Shiau's lab. The chemical structure and synthesis of TD19 has been disclosed in previous studies [31 (link),33 (link)], and its mechanisms as a SET inhibitor has been also reported in prior studies [30 (link),32 (link)]. Akt inhibitor (MK-2206) inhibitor and ERK inhibitor (SCH772984) were purchased from Selleckchem (Houston, TX, USA). TD19 at different concentrations were dissolved in dimethyl sulfoxide (DMSO) and added to cells in Dulbecco's Modified Eagle Medium (DMEM) containing 1% FBS. Antibodies used for Western blots of pElk-1 and Elk were obtained from Santa Cruz Biotechnology (San Diego, CA, USA), Other such as CIP2A, PARP, PP2Ac, Akt, pAkt (Ser473), beta-actin, caspase-3, ERK, pERK, pBcl2 (Ser70), Bcl2, pc-Myc (S62), c-Myc, Lamin B, Tubulin, GFP and Myc-tag were purchased from Cell Signaling (Danvers, MA, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich (St Louis, MO, USA). MTT was purchased from Sigma-Aldrich.
P c myc s62
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
P‐c‐Myc (S62) is a recombinant protein that corresponds to the phosphorylated form of the transcription factor c‐Myc at serine 62. It can be used as a standard or control in Western blotting experiments to detect and quantify the phosphorylation state of c‐Myc.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Cell Signaling Technology (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Pp2a c, by Cell Signaling Technology (1 mentions)
Cip2a, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Lamin b, by Cell Signaling Technology (1 mentions)
Erk inhibitor sch772984, by Selleck Chemicals (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Sf3b1, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Ppp2r5a, by Abcam (1 mentions)
Anti mouse igg hrp linked 7076, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
P p44 42 mapk, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
4 protocols using p c myc s62
1
Evaluation of SET Antagonist TD19 Effects
2
Quantitative Western Blot Protocol for Cellular Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses of whole‐cell protein lysates were performed essentially as reported previously [33 (link)]. In brief, protein lysates were quantified and separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes which were then probed with one of the following primary antibodies, SF3B1 (diluted at 1 : 2000; Abcam, Hanzhou, China, ab172634), c‐Myc (diluted at 1 : 1000; Cell Signaling Technology, Shanghai, China, #5605), p‐c‐Myc (S62) (diluted at 1 : 1000; Cell Signaling Technology, #13748), PPP2R5A (diluted at 1 : 2000; Abcam, ab89621), and β‐actin (diluted at 1 : 2000; Abcam, ab8226). Finally, membranes were supplemented with species‐specific secondary antibodies, and immunoreactivity was detected by Odyssey imaging system (LI‐COR Biosciences, Lincoln, NE, USA).
3
Mouse Mammary Epithelial Cell Lysate Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse mammary epithelial cell were lysed by RIPA buffer with protease inhibitor cocktail (25 955; Nacalai Tesque) and the whole‐cell lysate was obtained for IB analysis. Enhanced chemiluminescence reagent (WBKLS0500; Millipore, MA01821) was used to obtain the signals and visualize bands using the ImageQuant LAS4000 chemiluminescent image system. Quantification of the band intensity was undertaken using ImageJ‐NIH software (https://imagej.nih.gov/ij/ ). c‐Myc (9402), P‐c‐Myc‐(S62) (E1J4K) (13 748), p44/42 MAPK (9102), P‐p44/42 MAPK (T202/Y204), GSK‐3β (D5C5Z) XP(R) (12 456), P‐GSK‐3β (S9) (D85E12) (5558) XP(R), P‐Rb(S807/811) (9308), cyclin D1 (2926), PCNA (D3H8P) XP(R) (13 110), anti‐mouse IgG HRP‐linked (7076), and anti‐rabbit IgG HRP‐linked (7074) Abs were purchased from Cell Signaling Technology. Anti‐Myc (phospho‐Thr58) (Y011034), anti‐A‐tubulin mouse MAB (DM1A) and Rb (C‐15) sc:50 Abs were purchased from Applied Biological Materials, EMD Millipore, and Santa Cruz Biotechnology, respectively.
4
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in Pierce® RIPA buffer containing protease, and phosphatase inhibitor cocktail tablets (Roche, Mannheim, Germany). Equal amounts of protein lysate were resolved by SDS/PAGE and electrotransferred onto PVDF membranes. Membranes were processed according to standard procedure and proteins were detected using the ChemiDoc™ MP imaging system (Bio‐Rad, Hercules, CA, USA). Antibodies used for immunoblot analyses included antibodies for c‐Myc (Abcam, Cambridge, UK), G9a (Abcam), p‐c‐Myc (S62) (Cell Signaling Technology, Danvers, MA, USA), p‐c‐Myc (T58) (Cell Signaling Technology), H3K9me1 (Cell Signaling Technology), H3K9me2 (Cell Signaling Technology), H3K9me3 (Cell Signaling Technology), H3 (Cell Signaling Technology), Sox9 (Cell Signaling Technology), active β‐catenin (Merck Millipore, Temecula, CA, USA), total β‐catenin (BD Transduction, San Jose, CA, USA), p62 (Cell Signaling Technology), LC3B (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), caspase 3 (Cell Signaling Technology), PARP (Cell Signaling Technology) and β‐actin (Sigma).
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!