DOI: 1.138/n2131 Protein levels (% of ) e Full-length protein remining (%) 1 5 1 5 1 1 5 5 Hs7 Syt1 Syt2 β-atin CSP +tsyn Hs7 Syt1 Syt2 P4 rin.1.1.1 1 [Trypsin] (g/l) f +tsyn SNARE-omplexes remining (%) 1 8 6 4 2 1 8 6 4 2 Protein levels (% of ) +tsyn +tsyn 1 5 P4 rin β-atin CSP +tsyn +tsyn 4 6 8 1 Temperture ( C) +tsyn +tsyn g Remining fter hx hse (%) +tsyn +tsyn Protein reovere (% of ) 1 5 1 5 1 5 15 1 5 GDI 5 +tsyn SNARE levels omplexes 1 Protein reovere (% of ) ll 26 C 37 C 4 C 1 2 3 hx hse (h) P4 rin Remining protein levels fter hx hse (%) +tsyn 1 5 1 5 1 5 1 5 1 5 +tsyn +tsyn +tsyn ll 37 C Hs7 1 2 3 hx hse (h) Figure S1 Reue n SNARE omplex ssemly in KO mie.. Representtive lots (top) n summry grphs (ottom) of totl protein levels in rin homogentes otine from wil-type () n KO ( ) mie t P4. -interting proteins Hs7,, synptotgmin-1 (Syt1), n synptotgmin-2 (Syt2), s well s β-tin n were nlyze y quntittive immunolotting. Proteins were normlize to β-tin levels (n=4).. Sme s in (), exept rin homogentes from wil-type (), KO ( ), n KO mie expressing trnsgeni α-synulein ( +tsyn) were nlyze t P4 for Syntxin-1 (Synt- 1),, synptorevin-2 (), β-tin, n. Proteins were normlize to β-tin levels (n=4).. Representtive lots (top) n summry grphs (ottom) of immunopreipittions with rin homogentes otine from,, n +tsyn t P4 performe with ntioies to seletively reognize only monomeri.. Both the inputs n the immunopreipittes were immunolotte with ntioies to,, n ; GDI ws use s loing ontrol. Reovere protein (reltive to the input) ws first normlize to the immunopreipitte protein, n then to (n=4).. Sme s (), exept tht immunopreipittions were rrie out with ntioies to,, n tht reognize proteins oth in free form n fter ssemly into SNARE-omplexes. Representtive lots (left) n summry grphs (right) (n=3). e. Limite proteolysis of SNARE proteins in rin homogentes otine from, n +tsyn mie t P4. Brin homogentes were proteolyze for 1 min on ie using inite onentrtions of trypsin. Remining full-length protein ws mesure y quntittive immunolotting (for representtive immunolots, see Fig. 1) (n=5). f. KO ereses therml stility of SNARE-omplexes. Whole rin lystes from, n +tsyn mie (t P4) were inute in SDS smple uffer t inresing tempertures for 15 min. Smples were seprte y SDS-PAGE, n remining SDS-resistnt SNARE-omplexes were mesure y quntittive immunolotting. For representtive immunolots, see Fig. 1f (n=3). g. eletion estilizes in tempertureepenent mnner. Summry plots of turnover rtes of, Hs7,,,, n in ulture ortil neurons from littermte n, performe t inite tempertures (for immunolots, see Fig. 1h). Time ourse of protein egrtion ws mesure fter protein synthesis ws loke with.1 g/l yloheximie (hx). Cyloheximie hse experiments were strte t 9 ys in vitro (n=6). p<.5; p<.1; p<.1 using Stuent s t-test (in -) or one-wy ANOVA (in -f). www.nture.om/ntureelliology 1 21 Mmilln Pulishers Limite. All rights reserve.
Uiquitin TDP-43 + tsyn 36 Hs7 + tsyn + tsyn 36 Uiquitinte protein (%) 12 8 4 TDP-43 + tsyn 12 8 4 Hs7 + tsyn 12 8 4 + tsyn Figure S2 Uiquitintion of TDP-43, Hs7 n in KO mie. Anlysis of uiquitintion of TDP-43, Hs7, n in rins from mie, n KO mie without ( ) or with trnsgeni humn α-synulein ( +tsyn). Proteins soluilize in.1% Triton X-1 from rins of littermte mie t P4 were immunopreipitte using polylonl ntioies to the inite proteins, or pre-immune serum () ontrols ( ontrols shown). Boun protein n input (5% of totl) were immunolotte using monolonl ntioies to uiquitin (upper lots) or to the immunopreipitte proteins (lower lots). Signls from multiple inepenent experiments were mesure y quntittive immunolotting, n re plotte t the ottom for,, n +tsyn smples, normlize to the respetive non-uiquitinte protein immunopreipitte (omplementry to Fig. 2). p<.1 using Stuent s t-test; n=5. 2 www.nture.om/ntureelliology 21 Mmilln Pulishers Limite. All rights reserve.
CSP -/- C 2+ C 2+ Uiquitin Uiquitinte (%) 1 5 C 2+ 15 1 5 CSP -/- C 2+ Figure S3 Inrese uiquitintion of in KO mie is synpti tivity-epenent. Uiquitintion of ws mesure in neuronl ultures from n KO mie following inution (t DIV1) in ontrol meium (),.5 µm tetrootoxin (), or 4 mm C 2+ (C 2+ ), for 36 h () or 22 h ( KO) to ount for the estiliztion of SNAP- 25 in KO neurons. Neuronl proteins were soluilize in.1% Triton X-1 n immunopreipitte using polylonl ntioies to or pre-immune serum (; tretment shown). Immunopreipitte n input (5% of totl neuronl lyste) were immunolotte using monolonl ntioies to uiquitin (upper lots) or to (lower lots). Uiquitinte uner eh onition ws mesure y quntittive immunolotting n is shown t the ottom, normlize to the respetive non-uiquitinte protein immunopreipitte n to ontrol tretment. Dt re mens ± SEMs; p<.5; p<.1 using Stuent s t-test; n=3. www.nture.om/ntureelliology 3 21 Mmilln Pulishers Limite. All rights reserve.
levels (%) Remining protein (%) 16 12 8 4 1 5 1 5 hx (h) 5 1 15 2 2 2 2 2 Dys post-infetion (DPI) EGFP Hs7 1 2 3 8 16 24 36 Hs7 1 2 3 hx hse (h) EGFP- 1 2 3 EGFP EGFP- EGFP- SSPα EGFP 8 16 24 36 Remining protein (%) Hs7 EGFP 1 5 1 Hs7 5 2 4 2 4 hx hse (h) Figure S4, ut not its mutnt SSPα, stilizes n Hs7 in ulture neurons.,. Overexpression of EGFP- ut not EGFP-SSPα or EGFP lone inreses the levels of n Hs7, ut not of, syntxin-1 (), or synptorevin-2 (). Culture ortil neurons were infete with lentiviruses expressing EGFP only, EGFP-my-, or EGFP-my-SSPα t DIV2, n nlyze t inite ys post-infetion y quntittive immunolotting. Pnels show summry plots illustrting levels of,, Hs7, syntxin-1 (), n synptorevin-2 () t inite ys post infetion (; n=4), s well s the ely in protein egrtion upon yloheximie (hx) hse eginning t DIV16, inue y elevte levels of in neurons (; for representtive immunolots, see Fig. 4; n=5).,. Lk of ny effet of EGFP overexpression lone on the stility of synpti proteins in neurons. Mouse ortil neurons were infete with lentiviruses expressing n empty vetor (ontrol) or EGFP t DIV4, n sujete to hx hse experiment t DIV18. The mount of remining,,, n Hs7 t given time-points ws mesure y quntittive immunolotting (n=3). All t re mens ± SEMs; p<.5; p<.1 using Stuent s t-test (in ) or one-wy ANOVA (in ). 4 www.nture.om/ntureelliology 21 Mmilln Pulishers Limite. All rights reserve.
Hs7 α-syn VCP Coomssie IB: ATP ADP ATPγS 5% GST GST-Hs7 ADPβS ATP +ATPγS GST- GST- 11 ADP ATPγS ADPβS 5% GST GST-Hs7 5 5 1 5 2 1 2 1 % Protein Reovere in Immunopreipitte1 ATP ATPγS +ADPβS GST- GST- 11 ADP ADPβS Hs7 Figure S5 Hs7 ins to in n ADP-epenent mnner.. Hs7 ins to in n ADP-epenent mnner. Representtive lots (left) n summry grphs (right) of the effets of enine nuleoties on -epenent protein omplexes. Brin homogentes from littermte n KO mie ( ) t P4 were soluilize in.1% Triton X-1, supplemente with inite enine-nuleoties (1 mm) n 1 mm Mg 2+, n immunopreipitte with monolonl ntioies. The reovery of inite proteins ws nlyze y quntittive immunolotting, n is normlize for the immunopreipittion effiieny (n=4).. GST pullowns of purifie y GST lone, GST-Hs7, GST- or GST- in the presene of ATPγS (left) or ADPβS (right). GST inputs were nlyze using Coomssie rillint lue stining (top pnels), n pture ws ssesse y immunolotting (ottom pnels). For the reverse ining experiment, see Fig. 5. www.nture.om/ntureelliology 5 21 Mmilln Pulishers Limite. All rights reserve.
Hs7 remining (%) SNARE omplex formtion (%) 1 8 6 4 2 12 1 6 12 18 24 3 36 42 48 Inution t 37 o C (h) 8 6 4 2 6 12 18 24 3 36 42 48 Inution t 37 o C (h) + + + Hs7 + Hs7 + + Figure S6 /Hs7/ omplex hperones n enhnes its inorportion into SNARE omplexes in vitro.. Coomssie-stine SDS gels of purifie, Hs7, n.. Purifie (1 g/l) ws inute t 37 C with ifferent omintions of purifie, n Hs7, ll t equimolr onentrtion in.1 mm Mg 2+ -ATP. Aliquots were ollete t inite time points, n.1 mm Mg 2+ -ATP ws replenishe t the sme time for those smples tht ontinue. Dispperne of monomeri ws etete y SDS-PAGE n immunolotting (see Fig. 6 for representtive immunolots), n ws quntifie reltive to input levels. Inset inites tht the smple ontining ll three hperone omponents (Hs7, CSP, n ) is sttistilly signifintly ifferent (p<.1) from oth the smples ontining SNAP- 25 lone, or ontining plus Hs7 (see Fig. 6 for further quntittions; n=5).. Coomssie-stine SDS gels of purifie syntxin-1 () n synptorevin-2 ().. ontining smples from () were inute with equimolr mounts of syntxin-1 () n synptorevin-2 () t 4 C for 16 h. SNARE omplex formtion ws mesure y quntittive immunolotting using syntxin-1 ntioy (see Fig. 6 for representtive immunolots; n=5). p<.5; p<.1; p<.1 using Stuent s t-test (in ) or one-wy ANOVA (in n ). 6 www.nture.om/ntureelliology 21 Mmilln Pulishers Limite. All rights reserve.
f h Hs7 (A93) (CHAT33) (P4) Figure 1 Figure 2 11 11 Uiquitin (P4D1) (HPC1) (Cl 69.1) CSP (R) Hs7 (A93) Hs7 (A93) (CHAT33) (P4) (Cl 69.1) Figure 3 Figure 4 (HPC1) 11 11 Hs7 (Cl 3C5) -Syn (61786) Figure 5 Figure 6 (HPC1) 11 Figure S7 Full sns of key Western lot t. In mny experiments, memrnes were ut prior to proing eh strip with seprte ntioy. In these ses, the re etween two moleulr mss mrkers, t lest one ove n one elow the n is shown. Antioy use is inite in prentheses. www.nture.om/ntureelliology 7 21 Mmilln Pulishers Limite. All rights reserve.