1 MTSTGQDSTT TRQRRSRQNP QSPPQDSSVT SKRNIKKGAV PRSIPNLAEV KKKGKMKKLG 61 QAMEEDLIVG LQGMDLNLEA EALAGTGLVL DEQLNEFHCL WDDSFPEGPE RLHAIKEQLI 121 QEGLLDRCVS FQARFAEKEE LMLVHSLEYI DLMETTQYMN EGELRVLADT YDSVYLHPNS 181 YSCACLASGS VLRLVDAVLG AEIRNGMAII RPPGHHAQHS LMDGYCMFNH VAVAARYAQQ 241 KHRIRRVLIV DWDVHHGQGT QFTFDQDPSV LYFSIHRYEQ GRFWPHLKAS NWSTTGFGQG 301 QGYTINVPWN QVGMRDADYI AAFLHVLLPV ALEFQPQLVL VAAGFDALQG DPKGEMAATP 361 AGFAQLTHLL MGLAGGKLIL SLEGGYNLRA LAEGVSASLH TLLGDPCPML ESPGAPCRSA 421 QASVSCALEA LEPFWEVLVR STETVERDNM EEDNVEESEE EGPWEPPVLP ILTWPVLQSR 481 TGLVYDQNMM NHCNLWDSHH PEVPQRILRI MCRLEELGLA GRCLTLTPRP ATEAELLTCH 541 SAEYVGHLRA TEKMKTRELH RESSNFDSIY ICPSTFACAQ LATGAACRLV EAVLSGEVLN 601 GAAVVRPPGH HAEQDAACGF CFFNSVAVAA RHAQTISGHA LRILIVDWDV HHGNGTQHMF 661 EDDPSVLYVS LHRYDHGTFF PMGDEGASSQ IGRAAGTGFT VNVAWNGPRM GDADYLAAWH 721 RLVLPIAYEF NPELVLVSAG FDAARGDPLG GCQVSPEGYA HLTHLLMGLA SGRIILILEG 781 GYNLTSISES MAACTRSLLG DPPPLLTLPR PPLSGALASI TETIQVHRRY WRSLRVMKVE 841 DREGPSSSKL VTKKAPQPAK PRLAERMTTR EKKVLEAGMG KVTSASFGEE STPGQTNSET 901 AVVALTQDQP SEAATGGATL AQTISEAAIG GAMLGQTTSE EAVGGATPDQ TTSEETVGGA 961 ILDQTTSEDA VGGATLGQTT SEEAVGGATL AQTTSEAAME GATLDQTTSE EAPGGTELIQ 1021 TPLASSTDHQ TPPTSPVQGT TPQISPSTLI GSLRTLELGS ESQGASESQA PGEENLLGEA 1081 AGGQDMADSM LMQGSRGLTD QAIFYAVTPL PWCPHLVAVC PIPAAGLDVT QPCGDCGTIQ 1141 ENWVCLSCYQ VYCGRYINGH MLQHHGNSGH PLVLSYIDLS AWCYYCQAYV HHQALLDVKN 1201 IAHQNKFGED MPHPH

Fig S1. Identification of HDAC6 acetylation sites. The mass spectrometry analysis of in vitro acetylated HDAC6 for acetylation sites was performed twice with five different digestions (trpsin, chymotrypsin, V8DE, V8E and ArgC). Overall coverage was 86% and lysine coverage was 93%. Yellow: covered sequences; green: acetylated lysine; red: unacetylated lysine; grey: undetermined lysine.

Fig S1

A B

D E

Fig S2. HDAC6 alignement between species. Acetylation clusters A,B,C and D are more conserve between cattle, mouse and human HDAC6 than cluster E . The red star shows high conservation site, the blue star shows low conservation site.

C

Fig S2

*

Dea

cety

lase

Act

ivity

(CM

P)

WT A B C D E AB ABCD ABCDE 100 200 500 1000

BSA(ng)

Coomassie brilliant blue staining of recombinant proteins

Fig S3. A. Recombinant HDAC6 and mutant proteins purified from baculovirus infected insect cells. B. Histone deacetylase activity of wild type and mutant HDAC6 proteins.

A

B

Fig S3