Table 1. PCR primer sets.
| Primer pair | Plasmid | Method | FORWARD 5’ sequence | REVERSE 3’ sequence |
|---|---|---|---|---|
| HsEPS15 315- >*FORWARD QC/HSEPS15 315- >*REVERSE QC |
Hs EPS15 (1-314) pGEX-5X-1 | QuikChange mutagenesis | 5’-ATGATTCCACCATCAGACA GGGCCTGATTACAAAAGAAC ATCATAGG-3’ | 5’-CCTATGATGTTCTTTTGTAATC AGGCCCTGTCTGATGGTGGAAT CAT-3’ |
| EH2 W218* QC FORWARD/EH2 W218* QC REVERSE | Hs EPS15 (1-217) pGEX-5X-1 | QuikChange mutagenesis | 5’-CCATCTAAGAGAAAAACG TGAGTTGTATCCCCTGCAG-3’ | 5’-CTGCAGGGGATACAACTCAC GTTTTTCTCTTAGATGG-3’ |
| EH1 L110* QC FORWARD/EH1 L110* QC REVERSE | Hs EPS15 (1-109) pGEX-5X-1 | QuikChange mutagenesis | 5’-CCAAGATTTCATGATACCA GTAGTCCTTAACTAATCAGTG GAACCTCTGC-3’ | 5’-GCAGAGGTTCCACTGATTAGT TAAGGACTACTGGTATCATGAAA TCTTGG-3’ |
| Hs EPS15 Pro121 Phusion FORWARD/pGEX-5X-1 BamHI Phusion REVERSE |
Hs EPS15 (121-314) pGEX-4T-1 | Phusion mutagenesis | 5’-phos-CCATGGGCTGTA AAACCTGAAGATAAGG-3’ | 5’-phos-GATCCCACGACCTT CGATCAGATC-3’ |
| EPS15 W57A QC FORWARD/EPS15 W57A QC REVERSE | Hs EPS15 (1–107/W57A) pGEX-5X-1 | QuikChange mutagenesis | 5’-GACTTGATACTTGGAA AGATTGCGGATTTAGCCGAC ACAGATGGC-3’ | 5’-GCCATCTGTGTCGGCTAA ATCCGCAATCTTTCCAAGTATCA AGTC-3’ |
| Hs EPS15L1 EH1 BamHI-Met1 FORWARD/Hs EPS15L1 EH3 T366* XhoI REVERSE |
Hs EPS15R (1-365) pGEX-5X-1 | Directional cloning | 5’-GTGGGATCCTCACCAT GGCGGCGCCGCTCATCC-3’ | 5’-ATACTCGAGTTAGCCTCT CTCCGAAGGCGGGACCATGTCC −3’ |
| Primer A6E/Primer PCMF | Nb E_3-His6 pHEN6c |
Directional cloning | 5’-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3’ | 5’-CTAGTGCGGCCGCTGAG GAGACGGTGACCTGGGT-3’ |
| Primer A6E/Primer PCMF | Nb E-_142-His6 pHEN6c |
Directional cloning | 5’-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3’ | 5’-CTAGTGCGGCCGCTGAG GAGACGGTGACCTGGGT-3’ |
| Primer A6E/Primer PCMF | Nb E_180-His6 pHEN6c |
Directional cloning | 5’-GATGTGCAGCTGCAG GAGTCTGGRGGAGG-3’ | 5’-CTAGTGCGGCCGCTGAG GAGACGGTGACCTGGGT-3’ |
| NPF-APA QC primer FORWARD/NPF- APA QC primer REVERSE |
Nb E-_142 (NPF APA)-His6 pHEN6c |
QuikChange mutagenesis | 5’-TGTAATATGAAGAGCAT GGCCCCCGCCAGACGGTAT GACGTGTGG-3’ | 5’-CCACACGTCATACCGTCT GGCGGGGGCCATGCTCTTCATA TTACA-3’ |
| AgeI mNeonGreen 5' FORWARD/ XhoI mNeonGreen 3' REVERSE |
mNG pmNG-C1 | Directional cloning | 5’-GCGCTACCGGTCGCCACC ATGGTGAGCAAGGGCGAGG AGG-3’ | 5’-TTGAGCTCGAGATCTGAGTC CGGACTTGTACAGCTCGTCCAT GCCCATCAC-3’ |
| Nb 5’ BglII + Gly4 FORWARD/Nb 3’ HindIII REVERSE | mNG-Nb E_3 pmNG-C1 |
Directional cloning | 5’-ATCAGATCTGGAGGTGGA GGTCAGGTGCAGCTGCAGG AG-3’ | 5’-CTAAAGCTTTTATGAGGAGA CGGTGACCTGGG-3’ |
| Nb 5’ BglII + Gly4 FORWARD/Nb 3’ HindIII REVERSE | mNG-Nb E_142 pmNG-C1 |
Directional cloning | 5’-ATCAGATCTGGAGGTGGA GGTCAGGTGCAGCTGCAGG AG-3’ | 5’-CTAAAGCTTTTATGAGGAGA CGGTGACCTGGG-3’ |
| CORRECT_3 × 1–2 Nb E_142 BmsBI + Gly6Ser2 FORWARD/ |
mNG-Nb E_142 × 3 pmNG-C1 | Directional cloning | 5’-CACCGTCTCCTCATCCGG AGGTTCAGGAGGTTCCGGG GGCCAGGTGCAGCTGCAGG AG-3’ | 5’-GCCGTCGACTGAGGAGACG GTGACCTG-3’ |
| 3 × 2–3 Nb E_142 SalI + Gly6Ser2 FORWARD/BamHI 3x EPS142 Nb REVERSE for mNG-C1 |
mNG-Nb E_142 × 3 pmNG-C1 | Directional cloning | 5’-CTCAGTCGACGGCGGATC TGGTGGGAGTGGGGGACAG GTGCAGCTGCAGGAGTC-3’ | 5’-GGTGGATCCTTATGAGGAGA CGGTGACCTG-3’ |
| Nb 5’ BglII + Gly4 FORWARD/2X Nb E_142 QC primer HindIII-SalI REVERSE |
mNG-Nb E_142 × 3 pmNG-C1 | Directional cloning | 5’-ATCAGATCTGGAGGTGGA GGTCAGGTGCAGCTGCAGG AG-3’ | 5’-CCGCCGTCGACTGCACGAAA TTCGAAGCTTGAGGAGACGGTG ACCTGGGT-3’ |
| QC GFP-FYVE2- UbDelGG EcoRI sto/QC GFP- FYVE2-UbDelGG EcoRI sto |
GFP-FYVE2 pEGFP-C1 | QuikChange mutagenesis | 5’-GAGCAGCTGAACAAGAAG GCATAATTCATGCAGATCTTT GTG-3’ | 5’-CACAAAGATCTGCATGAATTA TGCCTTCTTGTTCAGCTGCTC-3’ |
| VHH Nb EcoRI +Gly4 forward/Anti-Eps15 VHH Nb ApaI reverse |
GFP-FYVE2-Nb E_142 pEGFP-C1 |
Directional cloning | 5’-ATCGAATTCGGAGGTG GAGGTCAGGTGCAGCTGCA GGAG-3’ | 5’-ACTGGGCCCTTATGAGGA GACGGTGACCTG-3' |
| VHH Nb EcoRI +Gly4 forward/3 maj Anti-Eps15 VHH Nb ApaI reverse |
GFP-FYVE2-dNb E_142 pEGFP-C1 |
Directional cloning | 5’-ATCGAATTCGGAGGTGGA GGTCAGGTGCAGCTGCAGG AG-3' | 5’-ACTGGGCCCTTAGAAGGA GACGGTGACCTG-3’ |
| Nb + NLS C-term Phusion FORWARD/Nb C- term Phusion REVERSE | GFP-Nb E_142- SV40 NLS pEGFP-C1 | Phusion mutagenesis | 5’-phos-GGACCCAAGAAGAAA CGGAAGGTGTGAGGGCCCG GGATCCACCG-3’ | 5’-phos-GCGGCCGCCTGAGGA GACGGTGACCTGGGTCC-3’ |
| MfeI EPS Nb142 FORWARD for GNbE3/BglII EPS15 Nb142 REVERSE for GNbE3 | G-Nb E_142-E3 pCAG_EGFP | Directional cloning | 5’-TCGAGCAATTGATGGCAGA AGTTCAGGTGCAGCTGCAGG AG-3’ | 5’-GGTAAGATCTTCCTGAGGAG ACGGTGACCTGGGTCCCCTGG CCC-3’ |