Table 1. Primers used for cloning of full-size or deletion mutants of SLAMF1, TRAM, TLR4, and Rab11 FIP2.
| Construct | For/Rev | Primer sequence (5′–3′) | Restriction enzyme | PCR product mapped on nucleotide sequence |
|---|---|---|---|---|
| SLAMF1 ( NM_003037.3 ) | ||||
| pcDNA3.1 and pLVX-EF1α-IRES-ZsGreen1 | For | TTCGAATTCTGATGGGATCCCAAGGGGCTCC | EcoRI | 367–1,374 |
| Rev | GCAGCGGCCGCTCAGCTCTCTGGAAGTGTCA | NotI | ||
| C-terminal DYKDDDDK | For | TTAGAATTCATGGATCCCAAGGGGCTCC | EcoRI | 367–1,371 |
| Rev | GGTACCTCGAGAGCTCTCTGGAAGTGTCACAC | XhoI | ||
| pGEX-2TK | For | TATGGATCCCAGTTGAGAAGAAGAGGTAAAACG | BamHI | 1,141–1,374 |
| Rev | ATAGAATTCTCAGCTCTCTGGAAGTGTCAC | EcoRI | ||
| SLAMF1 ( NM_003037.3 ) deletion mutants to C-terminal DYKDDDDK | ||||
| All deletion mutants | For | TTAGAATTCATGGATCCCAAGGGGCTCC | EcoRI | N/a |
| 1–265 aa | Rev | GGTACCTCGAGATTTACCTCTTCTTCTCAACTGTAG | XhoI | 367–1,161 |
| 1–326 aa | Rev | GTACCTCGAGAGACTGTGATGGAATTTGTTTCCTG | XhoI | 367–1,344 |
| 1–330 aa | Rev | GTACCTCGAGACACACTAGCATAGACTGTGATG | XhoI | 367–1,356 |
| SLAMF1 ( NM_003037.3 ) deletion mutant to pcDNA3.1 | ||||
| 1–265 aa | For | TTCGAATTCTGATGGGATCCCAAGGGGCTCC | EcoRI | 367–1,161 |
| Rev | TTAAGCGGCCGCTCATTTACCTCTTCTTCTCAACTG | NotI | ||
| SLAMF1 ( NM_003037.3 ) mutants with amino acid substitutions to C-terminal DYKDDDDK | ||||
| SLAMF1 Y281F | For | TTAGAATTCATGGATCCCAAGGGGCTCC | EcoRI | 367–1,225 |
| Rev | GTTTCTGGACTTGGGCAAAGATCGTAAGGC | |||
| For | GCCTTACGATCTTTGCCCAAGTCCAGAAAC | 1,196–1,371 | ||
| Rev | GGTACCTCGAGATTTACCTCTTCTTCTCAACTGTAG | XhoI | ||
| SLAMF1 Y327F | For | TTAGAATTCATGGATCCCAAGGGGCTCC | EcoRI | 367–1,371 |
| Rev | GGTACCTCGAGAGCTCTCTGGAAGTGTCACACTAGCAAAGACTGTG | XhoI | ||
| SLAMF1 TNSI/PNPT | For | TTAGAATTCATGGATCCCAAGGGGCTCC | EcoRI | 367–1,341 |
| Rev | TGTGGTGGGGTTTGGTTCCTGGACAGACTCTGG | |||
| For | GAACCAAACCCCACCACAGTCTATGCTAGTGTGACACTTC | 1,324–1,371a | ||
| Revb | CTTGTCATCGTCGTCCTTG | |||
| TRAM/TICAM-2 ( NM_021649.7 ) | ||||
| C-terminal DYKDDDDK | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–1,147 |
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | ||
| TRAM deletion mutants to C-terminal DYKDDDDK | ||||
| 1–68 aa | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–646 |
| Rev | TTAACTCGAGCCATCTCTTCCACGCTCTGAGC | XhoI | ||
| 1–79 aa | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–679 |
| Rev | TTACCTCGAGAGAGGAACACCTCTTCTTCAGC | XhoI | ||
| 1–90 aa | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–712 |
| Rev | TTACCTCGAGATGTGTCATCTTCTGCATGCAATATC | XhoI | ||
| 1–100 aa | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–742 |
| Rev | TTACCTCGAGATAGCAGATTCTGGACTCTGAGG | XhoI | ||
| 1–120 aa | For | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | 443–802 |
| Rev | TTACCTCGAGACTGTCTGCCACATGGCATCTC | XhoI | ||
| 68–235 aa | For | CATGAATTCATGTTTGAAGAAGAAGCTGAA | EcoRI | 644–1,147 |
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | ||
| 68–176 aa | For | CATGAATTCATGTTTGAAGAAGAAGCTGAA | EcoRI | 644–970 |
| Rev | TTAACTCGAGCCAGGGGCCGCATGGGTATAACAG | XhoI | ||
| 158–235 aa | For | CATGAATTCATGAACTCCGTTAACAGGCAGC | EcoRI | 914–1,147 |
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | ||
| TLR4 ( NM_138554.4 ) | ||||
| C-terminal DYKDDDDK | For | CGGTCGACCGAGATCTCATGATGTCTGCCTCGCGCCTGG | N/a | 299–2,815 |
| Rev | CTTGTAGTCGCCGGTACCGATAGATGTTGCTTCCTGCCAATTG | N/a | ||
| Mus Musculus TRAM/Ticam-2 ( NM_173394.3 ) to EGFP-N1 | ||||
| 1–232 aa | For | ACTAAGCTTATGGGTGTTGGGAAGTCTAAAC | HindIII | 477–1,175 |
| Rev | ATATGGATCCCGGGCAATGAACTGTTTCTGCGAC | BamHI | ||
| M. Musculus Slamf1 ( NM_013730.4 ) from pDisplay vector to C-terminal DYKDDDDK | ||||
| 30–343 aa | For | TTGGAATTCGGCTTGGGGATATCCACCATGG | EcoRI | 183–1,127 |
| Rev | TATACCTCGAGAGCTCTCTGGCAGTGTCACACTG | XhoI | ||
| Rab11 FIP2 ( NM_014904.2 ) and deletion mutants to N-terminal DYKDDDDK | ||||
| All constructs | For | GCCCGAATTCGGCTGTCCGAGCAAGCCCAAAAG | EcoRI | |
| 1–512 aa | Rev | ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC | NotI | 446–1,980 |
| 1–327 aa | Rev | ATAGCGGCCGCTCATTCGCTGCTTTCTTCAAATGG | NotI | 446–1,429 |
| 1–290 aa | Rev | ATAGCGGCCGCTTACACAATGCTGTCAGGTTGG | NotI | 446–1,310 |
| 1–254 aa | Rev | ATAGCGGCCGCTTATCCGAGAAGATGTGTTTGACC | NotI | 446–1,199 |
| 1–192 aa | Rev | ATAGCGGCCGCTTAGTGAGTACTTGGAATGATTGC | NotI | 446–1,016 |
| Rab11a ( NM_004663.4 ) QL and SN mutants to pECFP-C1 | ||||
| 1–216 aa | For | ATCAGTCGACATGGGCACCCGCGACGAC | SalI | 128–145 |
| Rev | TTAAGGATCCTTATATGTTCTGACAGCACTG | BamHI | 758–774 |
For, forward; QL, Rab11a Q70L mutant; Rev, reverse; SN, Rab11a S25N mutant.
Partially on vector sequence.
On C-terminal DYKDDDDK vector.