TABLE 1.
Strains and plasmids
| Strain or plasmid | Source and/or reference | Genotype or description |
|---|---|---|
| Strains | ||
| W303 | M. Rosbash (20) | MATa ade2-1 ura3-1 trp1.1 leu2-3,112 his3-11,15 |
| crm1.1 | L. Davis (20, 32) | MATa crm1-1 ade2 ura3 trp1 leu2 his3 |
| CRM1+ | This study | MATa crm1-1 ade2 ura3 trp1 leu2 his3; pLDB391 (CRM1 LEU2) integrated at CRM1 |
| EGY48 | M. Rosbash | MATα his3 ura3 trp13 lexA-op-LEU2; pSH18-34 (URA3 6lexA-op-lacZ) |
| RFY206 | M. Rosbash | MATa his3 leu2 trp1 lys2 ura3; pSH18-34 (URA3 6lexA-op-lacZ) |
| Yeast plasmids | ||
| pLDB391 | L. Davis (32) | XhoI-AatII fragment containing CRM1 ORF cloned into HindIII site (blunt ended) of pRS305 (LEU2) |
| pCGF-1A (pPS808) | P. Silver (13) | pPS293-based vector, 2μm, GFP under GAL promoter control, URA3, Ampr |
| pGFP | This study | pCGF-1A with terminator sequence from ADH in SalI and SphI sites |
| pGFP-IκBα | This study | Full-length human IκBα cDNA was cloned in frame after GFP in BamHI and XbaI sites of pGFP vector |
| pGFP-IκBαNEc | This study | Full-length human IκBα cDNA with leucine-to-alanine mutations at residues 272, 274, and 277 (L234) was cloned in frame after GFP |
| pGFP-NΔ32IκBα | This study | Human IκBα cDNA encoding residues from the number indicated after Δ (e.g., residue 32 in pGFP-NΔ32IκBα) to the end of the protein (residue 317) was cloned in frame after GFP |
| pGFP-NΔ42IκBα | ||
| pGFP-NΔ55IκBα | ||
| pGFP-NΔ59IκBα | ||
| pGFP-NΔ32IκBαNEc | This study | Same as above, except that the constructs contain L234 mutations at the C terminus |
| pGFP-NΔ42IκBαNEc | ||
| pGFP-NΔ55IκBαNEc | ||
| pGFP-NΔ59IκBαNEc | ||
| pGFP-NΔ71IκBαNEc | ||
| pGFP-NΔ84IκBαNEc | ||
| pGFP-IκBα-LIL3A49 | This study | Full-length human IκBα cDNA with leucine- or isoleucine-to-alanine mutations at residues 49, 52, and 54 was cloned in frame after GFP |
| pGFP-IκBαNEc-LIL3A49 | This study | Same as above, except that the construct contains L234 mutation at the C terminus |
| pGFP-IκBαNEc-LI2A78 | This study | Full-length human IκBα cDNA with leucine- or isoleucine-to-alanine mutations at residues 78, 82 (LI2A78), 272, 274, and 277 (L234) was cloned in frame after GFP |
| pGFP-IκBαNEc-LLII4A78 | This study | Full-length human IκBα cDNA with leucine- or isoleucine-to-alanine mutations at residues 78, 80, 82, 83 (LLII4A), 272, 274, and 277 (L234) was cloned in frame after GFP |
| pGFP-IκBβ | This study | Full-length mouse IκBβ cDNA was cloned in frame after GFP in BamHI and XbaI sites of pGFP vector |
| pGFP-IκBɛ | This study | Full-length mouse IκBɛ cDNA was cloned in frame after GFP in BamHI and XbaI sites of pGFP vector |
| pGFP-p65 | This study | Full-length mouse p65 cDNA was cloned in frame after GFP in BamHI and XbaI sites of pGFP vector |
| p424 (pLDB229) | L. Davis | 2μm, GALL promoter, CYC1 terminator, TRP1, Ampr |
| pGAL1 | This study | GAL1 promoter of p424 was replaced with GAL1 promoter from pCGF-1A |
| pGAL1.HAIκBα | This study | An HA tag (YPYDVPDYA) was created in the 5′ primer that was used for cloning IκBα by PCR. The full-length HA-tagged IκBα cDNA was inserted in BamHI and EcoRI sites of pGAL1 |
| pJG 4-5 | M. Rosbash | 2μm, GAL promoter, ADH terminator, TRP1, Ampr; A cassette containing SV40 NLS, the acid blob B42, and the HA epitope tag was inserted after GAL promoter (NL-B42-HA) |
| pJG-Rev | M. Rosbash | Full-length Rev cDNA was inserted after NL-B42-HA cassette |
| pEG202 | M. Rosbash | 2 μm, ADH promoter, ADH terminator, HIS3, Ampr; a LexA DNA binding domain was inserted after ADH promoter |
| pEG202-CRM1 | M. Rosbash | Full-length cDNA encoding yeast CRM1 was cloned in frame after LexA |
| pJG-IκBα | This study | Full-length human IκBα was inserted in frame at EcoRI and XhoI sites after NL-B42-HA cassette |
| pJG-IκBαNEc5A | This study | Full-length human IκBα with leucine- or isoleucine-to-alanine mutations at residues 265, 269, 272, 274, and 277 was inserted after NL-B42-HA cassette |
| pJG-IκBαΔNEc | This study | Same as above, except that the C-terminal putative NES from residues 265 to 277 (IQQQLGQLTLE) was deleted |
| pJG-IκBα-LIL3A49 | This study | Full-length human IκBα with leucine-to-isoleucine-to-alanine mutations at 49, 52, and 54 (putative N-terminal NES) was cloned in frame after NL-B42-HA cassette |
| pJG-IκBαNEc-LIL3A49 | This study | Same as above, except that in addition to the mutations at the putative N-terminal NES, putative C-terminal NES was mutated (L234) |
| pJG-α60 | This study | A cDNA encoding human IκBα from amino acid residues 1 to 60 was inserted after NL-B42-HA cassette |
| pJG-α60-LIL3A49 | This study | Same as above, except that the putative N-terminal NES was mutated |
| pJG-α73 | This study | A cDNA encoding human IκBα from amino acid residues 1 to 73 was inserted after NL-B42-HA cassette |
| pJG-α73-LIL3A49 | This study | Same as above, except that the putative N-terminal NES was mutated |
| pJG-β56 | This study | A cDNA encoding mouse IκBβ from residues 1 to 56, right before the first ankyrin repeat was inserted after NL-B42-HA cassette |
| pJG-ɛ122 | This study | A cDNA encoding mouse IκBɛ from residues 1 to 122, right before the first ankyrin repeat was inserted after NL-B42-HA cassette |
| pYEX-BX | Clontech | 2μm, pCUP1, URA3, LEU2-d, Ampr |
| pYEX-BX-IκBα | This study | A HA-tagged, full-length human IκBα was inserted into BamHI and EcoRI sites of pYEX-BX, under the control of a copper-inducible promoter |
| pCu.IκBα | This study | A HindIII (fill in)-EcoRI fragment containing HA-IκBα and the copper-induced promoter was excised from pYEX-BX-IκBα and cloned into SacI (fill in) and EcoRI sites of p424 |
| Mammalian plasmids | ||
| pEGFP.C3 | Clontech | SV40 ori, pUC ori, pCMV, EGFP, SV40 poly(A), Ampr |
| pGFP-p65 | This study | Full-length mouse p65 cDNA was inserted in frame behind GFP into XhoI and EcoRI sites of pEGFP.C3 |
| pGFP-IκBα | This study | Full-length human IκBα cDNA was cloned in frame after GFP |
| pGFP-IκBαNEc | This study | Same as above, except that the construct contains L234 mutations |
| pGFP-IκBαNEc-LIL3A49 | This study | Same as above, except that the construct also contains the N-terminal mutations |
| pGFP-IκBα-LIL3A49 | This study | Same as above, except that the construct does not contain L234 mutations |
| pGFP-IκBβ | This study | Full-length mouse IκBβ cDNA was cloned in frame after GFP |
| pGFP-IκBɛ | This study | Full-length mouse IκBɛ cDNA was cloned in frame after GFP |
| pCDNA3 | Clontech | SV40 ori, ColE1 ori, pCMV, BGH poly(A), Ampr |
| pCDNA3.HA-IκBα | This study | Full-length human IκBα with the HA tag was cloned into pCDNA3 |
a
ORF, open reading frame; SV40, simian virus 40; BGH, bovine growth hormone; EGFP, enhanced GFP; ADH, alcohol dehydrogenase.