Co-immunoprecipitation of FKBP51 and GR

Co-immunoprecipitation (co-IP) was performed as previously described.^{48 (link)} Briefly, HeLa cells were transfected with 3 μg of FKBP51 and treated overnight with hydrocortisone (50 nm) and benztropine (10 μM). Cells were harvested in M-PER buffer, and lysates were incubated overnight in rabbit anti-GR antibody (Cell Signaling) at 4 °C. Magnetic protein A beads (Life Technologies) were incubated with samples for 4 h at 4 °C, followed by five washes and a denaturing elution. The resulting precipitates were subjected to SDS-PAGE.

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Sabbagh J.J., Cordova R.A., Zheng D., Marrero M.C., Lemus A., Li P., Baker J.D., Nordhues B.A., Darling A.L., Martinez-Licha C., Rutz D.A., Patel S., Buchner J., Leahy J.W., Koren J I.I.I., Dickey C.A, & Blair L.J. (2018). Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD. ACS chemical biology, 13(8), 2288-2299.

Publication 2018

rabbit anti-GR antibody Magnetic protein A beads

Anti antibody Benztropine Buffer Cells Co immunoprecipitation Fkbp51 Hela cells Hydrocortisone Protein a Rabbit Sds page

Corresponding Organization : USF Health Byrd Alzheimer's Institute

Other organizations : Technical University of Munich

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Variable analysis

independent variables

FKBP51 transfection
Hydrocortisone treatment (50 nm)
Benztropine treatment (10 μM)

dependent variables

Interaction between FKBP51 and glucocorticoid receptor (GR)

control variables

Cell line (HeLa cells)
Cell lysis buffer (M-PER buffer)
Incubation time (overnight for lysate-antibody incubation, 4 h for lysate-bead incubation)
Incubation temperature (4 °C)
Magnetic protein A beads (Life Technologies)
Rabbit anti-GR antibody (Cell Signaling)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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