Reagents used in this study
| Reagents . | |||||
|---|---|---|---|---|---|
| Antibodies | |||||
| Rabbit anti-xCENP-A | Aaron Straight, Stanford University | Milks et al., 2009 | |||
| Rabbit anti-xCENP-C | Aaron Straight, Stanford University | Milks et al., 2009 | |||
| Rabbit anti-xM18BP1 | Aaron Straight, Stanford University | Moree et al., 2011 | |||
| Rabbit anti-FLAG | Sigma-Aldrich | Cat# F7425; RRID: AB_439687 | |||
| Mouse anti-FLAG | Sigma-Aldrich | Cat#. F1804; RRID: AB_262044 | |||
| Rabbit anti-Myc | EMD Millipore | Cat# 06–549; RRID: AB_310165 | |||
| Mouse anti-myc, clone 4A6 | EMD Millipore | Cat# 06–549; RRID: AB_310165 | |||
| Mouse anti-V5 | EMD Millipore | Cat# R960-25; RRID: AB_2556564 | |||
| Mouse anti-Tubulin, clone DM1A | Sigma-Aldrich | Cat# T9026; RRID: AB_477593 | |||
| AlexaFluor-conjugated secondaries | Thermo Fisher Scientific | Various | |||
| AlexaFluor-conjugated secondaries | Jackson Immunoresearch | Various | |||
| Oligonucleotides | |||||
| Forward primer for making S220A mutation in xHJURP-3xFLAG | 5′-AGCTTCAAGGTAGCGCCCATGAAGTTC-3′ | ||||
| Reverse primer for making S220A mutation in xHJURP-3xFLAG | 5′-GAACTTCATGGGCGCTACCTTGAAGCT-3′ | ||||
| Forward primer for making S220D mutation in xHJURP-3xFLAG | 5′-AGCTTCAAGGTAGATCCCATGAAGTTC-3′ | ||||
| Reverse primer for making S220D mutation in xHJURP-3xFLAG | 5′-GAACTTCATGGGATCTACCTTGAAGCT-3′ | ||||
| Forward primer for making P221A mutation in xHJURP-3xFLAG | 5′-TTCAAGGTATCGGCCATGAAGTTCCGC-3′ | ||||
| Forward primer for making P221A mutation in xHJURP-3xFLAG | 5′-GCGGAACTTCATGGCCGATACCTTGAA-3′ | ||||
| Plasmids | |||||
| 6xMyc-xCENP-C in pCS2+ | ASP 867 | ||||
| Myc-xCENP-C aa1–380 in pCS2+ | ASP 1330 | ||||
| Myc-xCENP-C aa381 - 711 in pCS2+ | ASP 1928 | ||||
| Myc-xCENP-C aa712–1191 in pCS2+ | ASP 1920 | ||||
| Myc-xCENP-C aa1191–1400 in pCS2+ | ASP 1150 | ||||
| Myc-xM18BP1.S in pCS2+ | ASP 1643 | ||||
| Myc-xM18BP1.L in pCS2+ | ASP 1645 | ||||
| xHJURP-3xFLAG in pCS2+ | ASP 1867 | ||||
| xHJURP-3xFLAG aa1-205 in pCS2+ | ASP 2040 | ||||
| xHJURP-3xFLAG aa1-400 in pCS2+ | ASP 2041 | ||||
| xHJURP-3xFLAG aa1-500 in pCS2+ | ASP 2042 | ||||
| xHJURP-3xFLAG aa201-690 in pCS2+ | ASP 2043 | ||||
| xHJURP-3xFLAG aa401-690 in pCS2+ | ASP 2456 | ||||
| xHJURP-3xFLAG aa201-400 in pCS2+ | ASP 2436 | ||||
| xHJURP-3xFLAG aa201-361 in pCS2+ | ASP 2437 | ||||
| xHJURP-3xFLAG aa305-400 in pCS2+ | ASP 2438 | ||||
| xCENP-A-Myc + H4 in pST39 | ASP 3451 | ||||
| xCENP-C-3xFLAG in pCS2+ | ASP 3533 | ||||
| Myc-xM18BP1.S T166A + SANTA in pCS2+ | ASP 3843 | ||||
| xHJURP-3xFLAG S220D in pCS2+ | ASP 4250 | ||||
| xHJURP-3xFLAG S220A in pCS2+ | ASP 4251 | ||||
| xHJURP-3xFLAG P221A in pCS2+ | ASP 4252 | ||||
| xHJURP-3xFLAG aa1-225 in 1853 | ASP 4256 | ||||
| 3xFLAG-CENP-C R1210A in pCS2+ | ASP 4257 | ||||
| 3xFLAG-CENP-C R874A in pCS2+ | ASP 4258 | ||||
| 3xFLAG-CENP-C F1322A,F1393A in pCS2+ | ASP 4259 | ||||
| 3xFLAG-CENP-C F1322A in pCS2+ | ASP 4260 | ||||
| xHJURP-3xFLAG aa1-250 in pCS2+ | ASP 4264 | ||||
| xHJURP-3xFLAG aa1-300 in pCS2+ | ASP 4265 | ||||
| xHJURP-3xFLAG aa1-350 in pCS2+ | ASP 4266 | ||||
| Reagents . | |||||
|---|---|---|---|---|---|
| Antibodies | |||||
| Rabbit anti-xCENP-A | Aaron Straight, Stanford University | Milks et al., 2009 | |||
| Rabbit anti-xCENP-C | Aaron Straight, Stanford University | Milks et al., 2009 | |||
| Rabbit anti-xM18BP1 | Aaron Straight, Stanford University | Moree et al., 2011 | |||
| Rabbit anti-FLAG | Sigma-Aldrich | Cat# F7425; RRID: AB_439687 | |||
| Mouse anti-FLAG | Sigma-Aldrich | Cat#. F1804; RRID: AB_262044 | |||
| Rabbit anti-Myc | EMD Millipore | Cat# 06–549; RRID: AB_310165 | |||
| Mouse anti-myc, clone 4A6 | EMD Millipore | Cat# 06–549; RRID: AB_310165 | |||
| Mouse anti-V5 | EMD Millipore | Cat# R960-25; RRID: AB_2556564 | |||
| Mouse anti-Tubulin, clone DM1A | Sigma-Aldrich | Cat# T9026; RRID: AB_477593 | |||
| AlexaFluor-conjugated secondaries | Thermo Fisher Scientific | Various | |||
| AlexaFluor-conjugated secondaries | Jackson Immunoresearch | Various | |||
| Oligonucleotides | |||||
| Forward primer for making S220A mutation in xHJURP-3xFLAG | 5′-AGCTTCAAGGTAGCGCCCATGAAGTTC-3′ | ||||
| Reverse primer for making S220A mutation in xHJURP-3xFLAG | 5′-GAACTTCATGGGCGCTACCTTGAAGCT-3′ | ||||
| Forward primer for making S220D mutation in xHJURP-3xFLAG | 5′-AGCTTCAAGGTAGATCCCATGAAGTTC-3′ | ||||
| Reverse primer for making S220D mutation in xHJURP-3xFLAG | 5′-GAACTTCATGGGATCTACCTTGAAGCT-3′ | ||||
| Forward primer for making P221A mutation in xHJURP-3xFLAG | 5′-TTCAAGGTATCGGCCATGAAGTTCCGC-3′ | ||||
| Forward primer for making P221A mutation in xHJURP-3xFLAG | 5′-GCGGAACTTCATGGCCGATACCTTGAA-3′ | ||||
| Plasmids | |||||
| 6xMyc-xCENP-C in pCS2+ | ASP 867 | ||||
| Myc-xCENP-C aa1–380 in pCS2+ | ASP 1330 | ||||
| Myc-xCENP-C aa381 - 711 in pCS2+ | ASP 1928 | ||||
| Myc-xCENP-C aa712–1191 in pCS2+ | ASP 1920 | ||||
| Myc-xCENP-C aa1191–1400 in pCS2+ | ASP 1150 | ||||
| Myc-xM18BP1.S in pCS2+ | ASP 1643 | ||||
| Myc-xM18BP1.L in pCS2+ | ASP 1645 | ||||
| xHJURP-3xFLAG in pCS2+ | ASP 1867 | ||||
| xHJURP-3xFLAG aa1-205 in pCS2+ | ASP 2040 | ||||
| xHJURP-3xFLAG aa1-400 in pCS2+ | ASP 2041 | ||||
| xHJURP-3xFLAG aa1-500 in pCS2+ | ASP 2042 | ||||
| xHJURP-3xFLAG aa201-690 in pCS2+ | ASP 2043 | ||||
| xHJURP-3xFLAG aa401-690 in pCS2+ | ASP 2456 | ||||
| xHJURP-3xFLAG aa201-400 in pCS2+ | ASP 2436 | ||||
| xHJURP-3xFLAG aa201-361 in pCS2+ | ASP 2437 | ||||
| xHJURP-3xFLAG aa305-400 in pCS2+ | ASP 2438 | ||||
| xCENP-A-Myc + H4 in pST39 | ASP 3451 | ||||
| xCENP-C-3xFLAG in pCS2+ | ASP 3533 | ||||
| Myc-xM18BP1.S T166A + SANTA in pCS2+ | ASP 3843 | ||||
| xHJURP-3xFLAG S220D in pCS2+ | ASP 4250 | ||||
| xHJURP-3xFLAG S220A in pCS2+ | ASP 4251 | ||||
| xHJURP-3xFLAG P221A in pCS2+ | ASP 4252 | ||||
| xHJURP-3xFLAG aa1-225 in 1853 | ASP 4256 | ||||
| 3xFLAG-CENP-C R1210A in pCS2+ | ASP 4257 | ||||
| 3xFLAG-CENP-C R874A in pCS2+ | ASP 4258 | ||||
| 3xFLAG-CENP-C F1322A,F1393A in pCS2+ | ASP 4259 | ||||
| 3xFLAG-CENP-C F1322A in pCS2+ | ASP 4260 | ||||
| xHJURP-3xFLAG aa1-250 in pCS2+ | ASP 4264 | ||||
| xHJURP-3xFLAG aa1-300 in pCS2+ | ASP 4265 | ||||
| xHJURP-3xFLAG aa1-350 in pCS2+ | ASP 4266 | ||||