supportinginformations5.docVIP

Supporting information S5 Supplementary Methods Reagents. Proteasomal fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC, cat. P802), proteasomal inhibitor epoxomicin (cat. PI127), human purified 20S proteasome subunit (PW8720) and anti-20S proteasome core subunits (PW8155) were all purchased from Enzo Lif Sci, PA. Phosphatase and protease inhibitor cocktails were purchased from Sigma-Aldrich. The cardiomyocyte isolation kit was purchased from Cellutron. Antibodies against PKC(?(cat. sc-208), PKC(I (cat. sc-209), PKC(II (cat. sc-210), PKC( (cat. sc-213), PKC( (cat. sc-211), PKC( (cat. sc-214), HSP27 (cat. sc-1048), ubiquitinated proteins (cat. sc-8017) and GAPDH (cat. sc-137179) were from Santa Cruz Biotechnology, CA. Anti-soluble oligomer A11 (cat. AB9234) was from Millipore, MA. Caspase 3 (cat. 9662), anti-cleaved caspase 3 (cat. 9664), Ik(??cat. ?????, p53 (cat. 2524) and anti-serine and threonine phosphorylation antibodies were from Cell Signaling Technology, MA. Recombinant PKCs were purchased from Cell Signaling Technology, MA. Oxyblot kit detection was from Millipore (cat. S7150). Anti-mouse IgG, anti-rabbit IgG and peroxidase-linked species-specific antibodies were purchased from GE Heathcare, NJ. PKC( and PKC( siRNA were purchased from Applied Biosystem and Lipofectamine 2000 from Invitrogen. Bortezomib was from LC laboratories, MA (cat. B-1408). Peptide synthesis. (V?-???an (PKC-specific inhibitor??corresponding to amino acids 642-647 [QLVIAN]), (IV5-3 (a (IPKC-specific inhibitor, amino acids 646-651 [KLFIMN]), (IIV5-3 (a (IIPKC-specific inhibitor, amino acids 645-650 [QEVIRN]) [1] and (V1-2 (an εPKC-specific inhibitor, amino acids 14-21 [EAVSLKPT]) [2] were synthesized and conjugated to TAT47-57 carrier peptide (amino acids 47–57 [YGRKKRRQRRR]) via a disulfide bond between Cys residues at the N-terminus of each peptide [3] by American Peptide, Inc. (Sunnyvale, CA). The ability of TAT47-57 to deliver the peptides into all the