Figure 1. Systematic proteomic analysis of the…
Figure 1. Systematic proteomic analysis of the CRL network at steady-state
(A) TSCs for CRL…
Figure 1. Systematic proteomic analysis of the CRL network at steady-state(A) TSCs for CRL components associated with each bait are indicated by the heat map. Associated proteins are depicted within the heat map if the TSCs for the given protein were in excess of 3. For a complete list of interacting proteins see Table S1. (B-F) Schematic representation of proteins associated with CUL1(B), CUL2(C), CUL3(D), CUL4A or CUL4B(E) and CUL5(F). See also Figure S1.
Figure 2. CSN activity within lysates alters…
Figure 2. CSN activity within lysates alters the architecture of the CRL network
(A) TAP-CUL1…
Figure 2. CSN activity within lysates alters the architecture of the CRL network(A) TAP-CUL1 cells were lysed in the presence or absence of 2mM 1,10 o-phenanthroline (OPT) and analyzed by SDS-PAGE and immunoblotted with α-HA antibodies. (B) 293T cells were either untreated or treated with 1μM MLN4924 for 4 hours (MLN4924-C). Untreated cells were then lysed without OPT, with 1–10 OPT, 1–7 OPT, or 1–10 OPT with MLN4924 added to the lysis buffer (MLN4924-L). The extent of cullin neddylation was determined by immunoblotting. Arrows indicate the neddylated species. (C) LC-MS/MS analysis of the indicated immune complexes in the presence or absence of OPT. TSCs were normalized by bait TSCs. Associated proteins are depicted within the heat map if the TSCs for the given protein were in excess of 3 within any of the immune complexes. (D) Comparison of cullin TSCs within TAP-NEDD8 immune complexes with (red bars) or without OPT (blue bars) in the lysis buffer. (E-G) Bait normalized TSCs for COPS1(E), COPS5(F), or CAND1(G) associated with the indicated TAP-immune complexes with (red bars) and without OPT (blue bars) in the lysis buffer. Error bars: STDEV of duplicate measurements (*,** = pvalue < 0.05, 0.01, respectively, by Student’s t-test). See also Figure S2 and Table S2.
Figure 3. Rapid deneddylation of CRLs in…
Figure 3. Rapid deneddylation of CRLs in response to NAE inhibition by MLN4924
(A) 293T…
Figure 3. Rapid deneddylation of CRLs in response to NAE inhibition by MLN4924(A) 293T cells with or without 1μM MLN4924 (4h) treatment were lysed in the presence of OPT and the extent of neddylation of endogenous cullins was determined by immunoblotting. *indicates non-specific background band. (B) 293T cells expressing the indicated TAP-tagged proteins with or without 4h MLN4924 treatment were lysed in the presence of OPT and immunoblotted with the indicated antibodies. Bait complexes were immunoprecipitated with α-HA and immunoblotted with the indicated antibodies. (C) Complexes were immunoprecipitated with α-HA coupled resin and blotted with antibodies against CUL1, CUL5, and CUL4A. (D) TAP-NEDD8 expressing cells with or without 4h MLN4924 treatment were lysed in the presence of OPT. α-HA complexes were analyzed by LC-MS/MS and bait normalized TSCs for known CRL components are displayed. (E-H) Normalized TSCs for cullins (E), CSN subunits (F), CRL adaptor proteins (G), and the NEDD8 conjugation machinery (H) associated with TAP-NEDD8 with (red bars) or without (blue bars) MLN4924 treatment. Error bars: STDEV of duplicate measurements (*,** = pvalue < 0.05, 0.01, respectively, by Student’s t-test).
Figure 4. Acute NAE1 inhibition does not…
Figure 4. Acute NAE1 inhibition does not globally alter the CRL network
(A) Extracts from…
Figure 4. Acute NAE1 inhibition does not globally alter the CRL network(A) Extracts from 293T cells expressing the indicated proteins (with or without 4h MLN4924 treatment) were immunoprecipitated with α-HA and associated proteins were identified by LC-MS/MS. Bait-normalized TSCs for associated CRL components are shown. (B) The relative abundance of cullins associated with CSN6, DCUN1D1, COPS5, or CAND1 immune complexes with (red bars) or without (blue bars) MLN4924 treatment. (C) Normalized TSCs for COPS1, COPS5, or CAND1 associated with the indicated immune complexes with (red bars) and without MLN4924 (blue bars) treatment. (D) Extracts from 293T cells expressing the indicated proteins (with or without 4h MLN4924 treatment) were probed with antibodies against COPS5. Bait complexes were immunoprecipitated with α-HA and immunoblotted for COPS5. (E) Bait normalized TSCs for a subset of adaptor proteins associated with their cognate cullin with (red bars) and without MLN4924 (blue bars) treatment. Error bars: STDEV of duplicate measurements (*,** = pvalue < 0.05, 0.01, respectively, by Student’s t-test). See also Figure S3 and Table S3.
Figure 5. Multiplex AQUA for quantitative proteomics…
Figure 5. Multiplex AQUA for quantitative proteomics of the CRL network
(A) Schematic multiplex AQUA-based…
Figure 5. Multiplex AQUA for quantitative proteomics of the CRL network(A) Schematic multiplex AQUA-based workflow. TAP-CUL1 was immunoprecipitated, eluted, and digested with trypsin. After peptide desalting, 100 fmoles of heavy-labeled AQUA reference peptide library targeting the indicated CRL components were added prior to LC-MS analysis. The colored lines under each CRL component indicate the number of AQUA peptides for that particular protein utilized in this study. See also Table S6. (B) MS chromatogram showing a heavy reference peptide (black) and its corresponding endogenous light peptide (red) for NEDD8 (left) and CUL1 (right) before (top) and after (bottom) MLN4924 treatment present within TAP-CUL1 immune complexes. m/z values are shown together with the corresponding peptide sequence (heavy labeled amino acid in red). (C) The concentration of the indicated components within TAP-CUL1 immune complexes from 293T cells was determined using multiplex AQUA. The mole fraction of CUL1 was then calculated by the ratio of abundances of the individual components and CUL1 with (red bars) and without MLN4924 (blue bars) treatment. CSN represents the average mole fraction calculated from AQUA measurements against each of the CSN subunits. (D) The mole fraction of TAP-CUL1 expressed in Hela cells bound to individual CRL components with (red bars) and without MLN4924 (blue bars) treatment. Error bars: STDEV of duplicate measurements (*,** = pvalue < 0.05, 0.01, respectively, by Student’s t-test). See also Figure S4.
Figure 6. Quantitative proteomic analysis of neddylation…
Figure 6. Quantitative proteomic analysis of neddylation deficient CUL1 complexes and time course analysis of…
Figure 6. Quantitative proteomic analysis of neddylation deficient CUL1 complexes and time course analysis of CUL1 complexes with MLN4924 treatment(A) Bait normalized TSCs of selected CRL components associated with wildtype TAP-CUL1 (with or without 4h MLN4924 treatment), a CUL1K720R mutant, and dominant negative CUL1 (CUL1DN). (B) HA-immunoblot of lysates from cells stably expressing wildtype TAP-CUL1 (with or without 4h MLN4924 treatment) or TAP-CUL1K720R. (C) Normalized TSCs for CAND1 (left) and CSN subunits (right) present in wildtype untreated and MLN4924 treated TAP-CUL1, TAP-CUL1K720R, and TAP-CUL1DN immune complexes. (D) Normalized TSCs for a subset of F-box proteins present in wildtype untreated (blue bars) and MLN4924 treated (red bars) TAP-CUL1, TAP-CUL1K720R (green bars), and TAP-CUL1DN (purple bars) immune complexes. (E) Multiplex AQUA analysis showing the mole fraction of the indicated CUL1 associated proteins present in untreated (blue bars) and MLN4924 treated (red bars) TAP-CUL1, and TAP-CUL1K720R (green bars) HA-immune complexes. (F) Extracts from 293T cells expressing TAP-CUL1 (with or without 1μM MLN4924 treatment for 2, 4, 8, or 16 h) were either immunoblotted directly or α-HA immune complexes were probed with the indicated antibodies. *indicates non-specific background band. (G) (top) Multiplex AQUA analysis of TAP-CUL1 immune complexes from (F) showing the mole fraction of NEDD8 (blue bars), CAND1 (red bars), SKP1 (green bars), and CSN (purple bars) bound to CUL1 with increasing time of MLN4924 treatment. (bottom) Multiplex AQUA analysis of TAP-CUL1 immune complexes from (F) showing the mole fraction of BTRC (blue bars) and FBXW11 (red bars) bound to CUL1. Error bars: STDEV of duplicate measurements (*,** = pvalue < 0.05, 0.01, respectively, by Student’s t-test, comparison between untreated and MLN time points) See also Figure S5.
Figure 7. Application of multiplex AQUA for…
Figure 7. Application of multiplex AQUA for assessment of CRL occupancy
(A) Schematic diagram using…
Figure 7. Application of multiplex AQUA for assessment of CRL occupancy(A) Schematic diagram using the CUL1 CRL as an example to show how each of the 9 different assemblages are calculated using multiplex AQUA measurements. The formulas used to calculate the abundance of each fraction are depicted. (B) The contribution of each of the assemblages depicted in (A) to the total occupancy of TAP-CUL1 immune complexes with and without MLN4924 treatment. The colors correspond to the colored assemblages in A. (C) The occupancy of TAP-CUL4B complexes calculated as in A, except that DDB1 replaced SKP1. The ratio of DDB1 to CUL4B in NEDD8 immune complexes represents the ratio of DDB1 to the combined concentrations of CUL4A and CUL4B. The colors correspond to the colored fractions in A. (D) Multiplex AQUA analysis of the mole fraction contribution of each of the 7 cullins associated with TAP-COPS6 (left) or TAP-COPS5 (right) with or without MLN4924 treatment. (E) Multiplex AQUA analysis of the mole fraction contribution of each of the 7 cullins associated with TAP-CAND1 with or without MLN4924 treatment. Error bars: STDEV of duplicate measurements. (F) Refined model of CRL dynamicity.
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