hours after food deprivation hours after food deprivation

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1 Figure S.6 protein (fasted / control) p47 p97 Rpt Ufd hours after food deprivation mrn (fasted / control) C mrn (denervated / control) p97 ** Npl4 Ufd p hours after food deprivation * * p47 * Ufd * Ufd2 * p97 * days after denervation Fig. S. The mrn of p97 and its cofactors are increased d after denervation, while only the mrn of p97 increases during fasting.. Levels of p97 and its cofactors Ufd and p47 and the proteasomal subunit Rpt were measured by immunoblotting on extracts from mouse Gastrocnemius at different times after food deprivation. Equal amounts of protein were loaded per lane. Densitometry data of protein levels are plotted. Error bars indicate SEM. n = 5. -C. Expression levels of p97 and its cofactors Ufd/Npl4, p47 and Ufd2 were measured by Real-Time PCR on extracts from mouse Gastrocnemius at different times after food deprivation () or after cutting the sciatic nerve (C). Equal amounts of mrn were used. Individual samples were run in triplicates and expressed relative to control samples. Error bars indicate SEM. * p <.5, ** p <.5. n = 3.

2 Figure S2 non-silencing shrn shrn p97.2 p97 tubulin p97 / tubulin * C UNC45 E non-silencing shrn control p97 GFP shrn UNC45 shrn p97-28 D UNC45 / control control shrn p97 shrn UNC45 Fig. S2. The shrn against p97 or UNC45 partially reduce the expression of the corresponding protein. -D. Murine neuroblastoma () or C2C2 myoblasts (C) were transfected for 72h with plasmids expressing a non-silencing shrn or a shrn against p97 or UNC45, respectively. Tubulin and are used as loading controls. The shrn reduced the protein amount by 4% in both cases, as shown by quantitation of bands in SDS PGE (-D). Error bars indicate SEM. * p <.5. E. Lysates from adult muscles electroporated with a mock vector (control) or a plasmid expressing a shrn against p97 (that also expresses GFP) for 7 d were loaded. The great efficiency of electroporation of this muscle (indicated by the high expression of GFP) resulted in silencing of endogenous p97.

3 Figure S3 p97- GFP Hours S I S I S I S I S I S I S I carbonylated proteins - 88 Fig. S3. In myotubes, overexpression of p97 does not cause accumulation of oxidatively-damaged proteins. Fully differentiated myotubes were infected for the indicated times with an adenovirus expressing p97 or GFP. Soluble (S) and insoluble (I) fraction of these cells in lysis buffer with % Triton X, mm DTT and 2M Urea were prepared. The presence of carbonylated proteins in equal amount of cell proteins was assayed after derivitization with P-hydrazine (PH) and then immunoblotting with an anti-p-hydrazone antibody.

4 Figure S4 % of input IP: actin IP: IgG % of input IP: IP: IgG actin polyubiquitin conjugates C % of input IP: actin IP: IgG + ortz + ortz + ortz - 88 (long expo) (short expo) polyubiquitin conjugates + ortz + ortz + ortz TP actin Fig. S4. In myotubes, p97 binds actin or in an TP-dependent way and results in accumulation of actin or as ubiquitinated species. -C. Immunoprecipitation of endogenous actin () with and without TP in the lysis buffer (C) or () was performed from myotubes overexpressing p97 or p97 for 72h (in presence of DMSO or µm ortezomib for 3h). ctin,, polyubiquitinated proteins (FK) and were immunoblotted. s control, immunoprecipitation of the same samples with appropriate IgG was carried out.

5 Figure S5 p97- p97- mock Hours Tot 4E-P P-4E-P Thr 37/46 P-4E-P Ser 65 p7 S6Kinase P-p7 S6Kinase Thr 389 p97 p97 P-kt Ser 473 kt Fig. S5. p97 or p97 overexpression does not alter the kt/mtor pathway in myotubes. -. Equal amounts of protein lysates from myotubes expressing p97 or p97 for the indicated times () or for 48h () were loaded and blotted with antibodies against:, 4E-P (and its phosphorylated forms in Thr 37/46 or Ser 65), p7s6kinase (and its phosphorylated form in Thr 389), kt (and its phosphorylated form in Ser 473) and as loading control.

6 Figure S6 Hours p97 p p97- (long expo) Fig. S6. p97 causes accumulation of ubiquitinated in myotubes. In total extracts of myotubes expressing p97 or p97 for the indicated times, protein was assayed by immunoblotting. accumulated as ubiquitinated species upon 6h-expression of p97.

7 SUPPLEMENTRY MTERIL ND METHODS For the validation of shrn constructs, murine neuroblastoma (NE-5) or myoblasts (C2C2) were maintained in DMEM + % FS (Invitrogen) and transfected with shrn constructs using Lipofectamine 2 (Invitrogen). Cells were lysed 72h later, and immunoblotting was performed. Protein extraction and immunoblotting Cell lysates were prepared as indicated in Material and Methods. The following antibodies were used anti-tubulin (Rockland), anti-4e-p, anti-phospho-4e-p (Thr 37/46), anti-phospho-4e-p (Ser 65), anti-p7s6kinase, anti-phospho-p7s6kinase (Thr 389), anti-kt and anti-phospho-kt (Ser 473) (Cell Signaling). RN isolation, Reverse Transcription and Quantitative Real-Time PCR Total RN was isolated from muscles with TRIzol (Invitrogen). RN concentration, purity and integrity were measured in a spectrophotometer (Ultrospec 2, Pharmacia iotech) and by running a denaturing agarose gel. nalysis of mrn / µg in muscle was performed using TaqMan reverse transcription reagents (pplied iosystems). Real-Time PCR reactions were carried out with DyNmo HS SYR Green qpcr kit (Finnzymes) and the appropriate primer pairs (Supplementary Table II) in an I 79HT fast Real-Time PCR system (pplied iosystems). Supplementary Table II: Molecule Sense primer sequence (5-3 ) ntisense primer sequence (5-3 ) p97 CTGGCCTTCGTGTCT GCCTGGTCGTCCTCTTCT Npl4 CTGCTGGCCGTGGGC CCGGTTGGTTCTGGGT Ufd CCTCGCCTGGGCT CGGTGCTCGGTCTTTT p47 CTGGCCTGGGGGCG CTGTGCCTCTGGCCTG Ufd2 GTCGCTTGGGGCGG CGCGTGCGTGGGTG