Supporting Information

Supporting Information

Plasmid Construction DNA cloning was carried out according to standard protocols. The sequence of all plasmids was confirmed by DNA sequencing and amplified using the E. coli strain DH5α and the E. coli strain BL21 (DE3) was used as the protein production host. NLS-mCherry (pjhj004): The synthetic DNA oligo encoding the NLS (KRPAATKKAGQAKKKKLD) was inserted into the pet28a vector between NdeI and EcoRI sites. Then the PCR amplified mcherry gene using primer set 1 was inserted to the resulting vector between EcoRI and XhoI sites to create pjhj004 plasmid. His6-NLS N -VMA N -CaM (pkdh018): The PCR amplified VMA N gene using primer set 2 and His6-NLS N gene using primer set 3 (template: pjhj001) were sequentially inserted into the pet28a between NheI and SalI sites and between NcoI and NheI sites, respectively. The CaM gene was amplified from cdna library using primer set 4 and was inserted into the resulting vector between SalI and XhoI sites to create pkdh018 plasmid. CaMBD-VMA C -NLS C -mcherry-his6 (pjhj015): The PCR amplified VMA C gene using primer set 5 and NLS C - mcherry gene using primer set 6 (template: pjhj004) were sequentially inserted into the pet28a between NdeI and NheI sites and between NheI and XhoI, respectively. Synthetic CaMBD encoding gene was inserted to the resulting vector between NcoI and NdeI sites to create pjhj005 plasmid. Finally, the CaMBD-VMA C -NLS C - mcherry-his6 was amplified using the pjhj005 plasmid as a template using primer set 7 and was inserted into MCS2 of mammalian expression vector, pbi-cmv1, between ApaI and XbaI sites to create pjhj015 plasmid. His6-NLS N -VMA N -CaM and CaMBD-VMA C -NLS C -mcherry His6 (pkdh022): The PCR amplified His6- NLS N -VMA N -CaM gene (template: pkdh018, primer set 8) were inserted into MCS1 of the pjhj015 between MluI and EagI sites to create pkdh022 plasmid. mcherry-caax-npu N -NLS (pjhj067): The PCR amplified mcherry gene (template: pjhj004, primer set 9) was inserted into the pbi-cmv1 between EcoRI and ApaI sites to create pjhj066 plasmid. Synthetic CAAX- Npu N -NLS encoding gene was inserted to the resulting vector between ApaI and XbaI sites to create pjhj067 plasmid. mcherry-caax-npu N -NLS and mnpu C -GR (pjhj075): The PCR amplified mnpu C gene (template: pjhj063, primer set 10) was inserted into the pjhj067 between BamHI and MluI sites to create pjhj072 plasmid. The GR gene was amplified from Human Pituitary Gland QUICK-Clone cdna library using primer set 11 and was inserted into the resulting vector between MluI and EcoRV sites to create pkdh075 plasmid. Npu C -Flag-Flag-GST (pjhj062): A PCR amplified GST gene (template: pegx-4t-1, primer set 12), synthetic Flag tag oligomer, and a PCR amplified Npu C (template pjdh012, primer set 13) were sequentially inserted to pet28a vector in between EcoRI and SacI sites, between BamHI and HindIII sites, and between NcoI and Bam- HI sites, respectively to create pjhj062. 2

mnpu C -Flag-Flag-GST (pjhj063): The pjhj063 was prepared by the same approach as pjhj062 but with primer set 13 instead of primer set 14. mcherry-caax (pjhj057): The synthetic DNA oligo encoding the CAAX tag (KRPAATKKAGQAKKKKLD) was inserted into the pet28a vector between NdeI and EcoRI sites. Then the PCR amplified mcherry gene (template: pjhj004, primer set 15) was inserted to the resulting vector between EcoRI and XhoI sites to create pjhj056 plasmid. Finally, the mcherry-caax tag was amplified using the pjhj056 plasmid as a template using primer set 16 and was inserted into MCS1 of mammalian expression vector, pbi-cmv1, between MluI and EagI sites to create pjhj057 plasmid. mcherry-caax-rphr (pjhj061): The pjhj058 was prepared by the same approach as pjhj057 but with primer set 17 instead of primer set 16 mcherry-gr (pkcw004): The PCR amplified GR gene (template: Human Pituitary Gland QUICK-Clone cdna, primer set 18) was inserted into MCS1 of the pbi-cmv1 between BamHI and MluI sites to create pkcw001 plasmid. The PCR amplified mcherry gene using primer set 19 was sequentially inserted into the pkcw001 between MluI and EcoRV sites. # of primer set Primer name Sequence (5 to 3 ) 1 2 3 4 5 6 7 8 mcherry_ecori_f mcherry_xhoi_r VMA N _NheI_F VMA N _SalI_R His6_NcoI_F NLS N _NheI_R CaM_SalI_F CaM_XhoI_R VMAC_NdeI_F VMAC_NheI_R NLS C _NheI_F mcherry_xhoi_r CaMBD_ApaI_F His6_XbaI_R His6_MluI_F CaM_EagI_R GAATTCATGGTGAGCAAGGGCG CTCGAGTTACTTGTACAGCTCGTCCA AGTGCTCCATATGGTTTTGCTTAACGTTCT GCGGCAGCCATATGAAACGTCC CAATTCTTTATGGATCCGGTGG GCCACCAAGAAAGCTAGCTGCTTTG GTCGACCTGACCAACTGACTG CTCGAGTCACTTTGCTGTCATCATTTGT AGTGCTCCATATGGTTTTGCTTAAC TAACTGTTTCAACGCTAGCGGCCAG CACCGCCACCCATATGTGCGTAGTCTGG CTCGAGTTACTTGTACAGCTCGTCCA TAACTGTTTCAACGCTAGCGGCCAG GTTAGCAGCTCTAGATCAGTGGTGGTGGTG GTCAGCTGACGCGTATGGGCAGCAG CTACGCAGGATCCGGTGGCGGTG 9 mcherry_ecori_f GATCCGGCTAGCATGGACTCCAAAGAATC 3

10 11 12 13 14 15 16 17 18 19 mcherry_apai_r NpuI_C_BamHI_F NpuI_C_MluI_R GR_MluI_F GR_EcoRV_R GST_EcoRI_F GST_SacI_R Npu_C_NcoI_F Npu_C(NCFN)_BamHI_R2 Npu_C_NcoI_F Npu_C(AAFN)_BamHI_R2 mcherry_bamhi_f mcherry_mlui_r mcherry_mlui_f CAAX_EagI_R mcherry_mlui_f CAAX(StopX)_EagI_R GR_BamHI_F GR_MluI_R mcherry_mlui_f mcherry_ecorv_r CATCCGGCCGCATGCTGGGGC GGAGATATGGATCCATGATCAAAATAGCCACACG CTTTATAATCACGCGTATTGAAAGCAGCAGAAG GATCCGACGCGTATGGACTCCAAAGAATC CTCGAGGATATCTCACTTTTGATGAAACAGAAG GATAAAAAGCTTATGTCCCCTATAC GACCATCCTCCAAAATGACTCGAG CAAAACCATGGCTATCAAAATAGCCACACGTAA CCGGATCCATTGAAACAATTAGAAGC CAAAACCATGGCTATCAAAATAGCCACACGTAA CCGGATCCATTGAAAGCAGCAGAAGCTATG GCTGGGATCCATGGTGAGCAAGGGC CTTCTTACGCGTCTTGTACAGCTCGTCC GGATCCACGCGTATGGACTCCAAAGAATC CTCGAGGATATCTCACTTTTGATGAAACAGAAG GCTGGAATTCATGGTGAGCAAGGGC CTTCTTGGGCCCCTTGTACAGCTCGT GATCCGACGCGTATGGACTCCAAAGAATC CTCGAGGATATCTCACTTTTGATGAAACAGAAG GATCCGACGCGTATGGACTCCAAAGAATC GACAGCGATGGGCCCATGAACGAAC Protein sequence His6- NLS N -VMA N -CaM (1) Expected Mass (kda): 40.4 HHHHHHSSGLVPRGSHMKRPAATKKASCFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLP RGRETMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEVITFEMGQ KKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARDLSLLGSHVRKATYQTYAP ILYGSADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNP XEAELQDMINEVDXDGNGTIDF LEFLTMXXRKXKDTDSEEEIXEAFRVXDKXGNGYISXAEXXHVMTNLGEKLTDEEVDEMIREADXDGD GQVNYEEXVQMRQQSDSRALXASREMX CaMBD-VMA C -NLS C -mcherry-his6 (2) Expected Mass (kda): 40.2 4

MAAARKEVIRNKIRAIGKMARVFSVLHMVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKED DYYGITLSDDSDHQFLLANQVVVHNCFNASGQAKKKKLDEFMVSKGEEDNMAIIKEFMRFKVHMEGS VSGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGF KWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG WEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKSLEHHHHHH mnpu C -GR (3) Expected Mass (kda): 90.2 MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASAAFNASMDSKESLTPGREENPSSVLAQERGDVMD FYKTLRGGATVKVSASSPSLAVASQSDSKQRRLLVDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETK VMGNDLGFPQQGQISLSSGETDLKLLEESIANLNRSTSVPENPKSSASTAVSAAPTEKEFPKTHSDVSSEQ QHLKGQTGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGEDDSFLL EGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPG ANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTS LGTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRA VEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGN KTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNL HLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELH RLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDS MHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGNIKKLLFHQK mcherry-caax-npu N -NLS (4) Expected Mass (kda): 45.9 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ FMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIK LDITSHNEDYTIVEQHERAEGRHSTGGMDELYKGPKHKEKMSKDGKKKKKKSKTKCVIMCLSYETEILT VEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDG QMLPIDEIFERELDLMRVDNLPNIKIATRKYLGKQNVYDIGVERKRPAATKKAGQAKKKKLD 5

MBP-Npu N (5) Expected Mass (kda): 57.3 MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFG GYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKE LKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMN ADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKE LAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA VRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFCLSYETEILTVEYGLLPIGKIVE KRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFEREL DLMRVDNLPNIKIATRKYLGKQNVYDIGVER Npu C -Flag-Flag-GST (6) Expected Mass (kda): 31.4 MAIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCFNGSDYKDDDDKKLMSPILGYWKIKGLVQPT RLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCP KERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFML YDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK mnpu C -Flag-Flag-GST (7) Expected Mass (kda): 31.4 MAIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASAAFNGSDYKDDDDKKLMSPILGYWKIKGLVQP TRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGC PKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK mcherry-caax (8) Expected Mass (kda): 29.8 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ 6

FMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIK LDITSHNEDYTIVEQHERAEGRHSTGGMDELYKEFKHKEKMSKDGKKKKKKSKTKCVIM mcherry-caax-rphr (9) Expected Mass (kda): 30.4 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ FMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAK KPVQLPGAYNVNIK LDITSHNEDYTIVEQHERAEGRHSTGGMDELYKEFKHKEKMSKDGKKKKKKSKTKCVIMRPHR mcherry-gr (10) Expected Mass (kda): 112.6 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQ FMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSD GPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIK LDITSHNEDYTIVEQHERAEGRHSTGGMDELYKTRMDSKESLTPGREENPSSV LAQERGDVMDFYKTLR GGATVKVSASSPSLAVASQSDSKQRRLLVDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETKVMGNDL GFPQQGQISLSSGETDLKLLEESIANLNRSTSVPENPKSSASTAVSAAPTEKEFPKTHSDVSSEQQHLKGQ TGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGEDDSFLLEGNSNED CKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNK MSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFP GRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHN YLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPAT LPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQM TLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYE EYLCMKTLLLLSSVPKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVE NLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGNIKKLLFHQK 7

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