Supplementary Figure 5: Immunoblot and immunohistochemical quantiification of CRBN. | Nature Structural & Molecular Biology

Supplementary Figure 5: Immunoblot and immunohistochemical quantiification of CRBN.

From: Structure of the human Cereblon–DDB1–lenalidomide complex reveals basis for responsiveness to thalidomide analogs

Supplementary Figure 5

(a) Immunoblot analysis of CRBN protein in lysates from DF15, DF15R, DF15R RFP (RFP Ctrl), DF15R CRBNWT (CRBN wt), DF15R CRBN W386A and CRBN W400A cells. (b) CRBN analysis in DF15 and DF15R and DF15R derived cell lines by immunohistochemistry. Images were obtained using a Olympus BX45 microscope at a 40x objective. CRBN signal is shown as brown color and hematoxylin counterstain identifies the nucleus of cells. (c) Immunoblot of anti-Flag immunoprecipitation from cell extracts expressing Flag-tagged CRBN proteins. (d) Immunoblot of thalidomide analog affinity bead binding to CRBN in DF15, DF15R and DF15R CRBNWT cell extracts. Lane description in order left to right: In = DF15 input prior to bead purification; V = DF15 extract control (1% DMSO preincubation); L = DF15 extract preincubated with lenalidomide (30 μM); P = DF15 extract preincubated with Pomalidomide (30 μM); In = DF15R input prior to bead purification; V = DF15R control (1% DMSO preincubation); L = DF15R extract preincubated with lenalidomide (30 μM). P = DF15R extract preincubated with Pomalidomide (30 μM); In = DF15R CRBNWT input prior to bead purification; V = DF15R CRBNWT control (1% DMSO preincubation); L = DF15R CRBNWT extract preincubated with lenalidomide (30 μM). P = DF15R CRBNWT extract preincubated with Pomalidomide (30 μM); Representative immunoblot from two independent experiments with similar results.

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