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BioAssay: AID 602146

EGFR degradation assay in SCC-12F cells

Stimulation by EGF (Epidermal Growth Factor) results in the internalization of EGFR (Epidermal Growth Factor Receptor) and trafficked in a Rab7-depended manner to the lysosome and subsequently degraded. Thus compounds that affect the activity of Rab family GTPases would decrease the rate of EGFR degradation. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(5)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(5)
 
 
Inactive(1)
 
 
AID: 602146
Data Source: NMMLSC (EGFR_Degradation_SCC12Fcells_GTPaseCompounds)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-12
Hold-until Date: 2012-12-05
Modify Date: 2012-12-05

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 5
Depositor Specified Assays
AIDNameTypeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasessummarySummary report for GTPase target project
Description:
University of New Mexico Assay Overview:
Assay Support: NIH I RO3 MH081231-01
HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Implementation: Lin Hong, Ph.D.
Target Team Leader for the Center: Larry Sklar (lsklar@salud.unm.edu)
UNM Cheminformatics: Cristian Bologa, Ph.D., Oleg Ursu, Ph.D.

Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.

Assay Background and Significance:
Stimulation by EGF (Epidermal Growth Factor) results in the internalization of EGFR (Epidermal Growth Factor Receptor) and trafficked in a Rab7-depended manner to the lysosome and subsequently degraded. Thus compounds that affect the activity of Rab family GTPases would decrease the rate of EGFR degradation.
Protocol
For degradation assay, SCC-12F cells grown in a six-well plate are serum starved for 2 hours in DMEM containing 10-100 microM of compound and then stimulated with 20 nanoM EGF. At different time points (0-2hours), cells are lysed with SDS lysis buffer containing a cocktail of proteinase inhibitors. Cell debris is removed by centrifugation, and the total amount of proteins is quantified by BSA assay. The amounts of EGFR are analyzed by SDS-PAGE and Western blot.

Response Assessment:
Western blots bands were scanned and normalized to the initial time point. The time course of protein degradation in the presence of compound were compared with degradation in presence of DMSO. Based on the decrease rate of degradation compared with DMSO sample, the compounds were assesses as active. And based on the magnitude difference in this degradation rate, the PUBCHEM_ACTIVITY_SCORE were given. An active compound has a SCORE > 50.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AVERAGE_DSMO_0MIN (0μM**)Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MINFloat
2STDEV_DSMO_0MIN (0μM**)Standard deviation western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 0MINFloat
3AVERAGE_DSMO_15MIN (0μM**)Average western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MINFloat
4STDEV_DSMO_15MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 15MINFloat
5AVERAGE_DSMO_30MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MINFloat
6STDEV_DSMO_30MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 30MINFloat
7AVERAGE_DSMO_60MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MINFloat
8STDEV_DSMO_60MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 60MINFloat
9AVERAGE_DSMO_120MIN (0μM**)Average of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MINFloat
10STDEV_DSMO_120MIN (0μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of DMSO at time 120MINFloat
11AVERAGE_10uM_0MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MINFloat
12STDEV_10uM_0MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 0MINFloat
13AVERAGE_10uM_15MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MINFloat
14STDEV_10uM_15MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 15MINFloat
15AVERAGE_10uM_30MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MINFloat
16STDEV_10uM_30MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 30MINFloat
17AVERAGE_10uM_60MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MINFloat
18STDEV_10uM_60MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 60MINFloat
19AVERAGE_10uM_120MIN (10μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MINFloat
20STDEV_10uM_120MIN (10μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 10uM at time 120MINFloat
21AVERAGE_30uM_0MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MINFloat
22STDEV_30uM_0MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 0MINFloat
23AVERAGE_30uM_15MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MINFloat
24STDEV_30uM_15MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 15MINFloat
25AVERAGE_30uM_30MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MINFloat
26STDEV_30uM_30MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 30MINFloat
27AVERAGE_30uM_60MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MINFloat
28STDEV_30uM_60MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 60MINFloat
29AVERAGE_30uM_120MIN (30μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MINFloat
30STDEV_30uM_120MIN (30μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 30uM at time 120MINFloat
31AVERAGE_100uM_0MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MINFloat
32STDEV_100uM_0MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 0MINFloat
33AVERAGE_100uM_15MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MINFloat
34STDEV_100uM_15MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 15MINFloat
35AVERAGE_100uM_30MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MINFloat
36STDEV_100uM_30MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 30MINFloat
37AVERAGE_100uM_60MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MINFloat
38STDEV_100uM_60MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 60MINFloat
39AVERAGE_100uM_120MIN (100μM**)Average western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MINFloat
40STDEV_100uM_120MIN (100μM**)Standard deviation of western blot band for EGFR, normalized to 0 time in the presense of 100uM at time 120MINFloat

** Test Concentration.
Additional Information
Grant Number: NIH I RO3 MH081231-01

Data Table (Concise)
Classification
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