Anti lc3

Western blots were performed as previously described [28 (link)]. Spinal cord and gastrocnemius tissue samples were disaggregated using Direct Quant 100ST Buffer (DireCt Quant) and a G50 Tissue Grinder (Coyote Bioscience). Total cell lysates of cultured cells or tissue homogenates were resolved in SDS polyacrylamide gels and transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters (Millipore), using an Amersham Biosciences semidry Trans-blot (Buckinghamshire, UK). The membranes were blotted with anti-SMN (1:5000; Cat. No. 610646, BD Biosciences), anti-LC3 (1:1000; Cat. No. 2775), anti-BECLIN-1 (1:1000; Cat. No. 3738), anti-p62/SQSTM1 (1:1000; Cat. No. 5114), anti-LAMP-1 (1:1000; Cat. No. 3243), anti-p-mTOR (1:1000; Cat. No. 5536) all from Cell Signaling Technology). To control the specific protein content per lane, membranes were reprobed with anti-CypA (1:10,000; Cat. No. BML-SA296-0100, Enzolifesciences) or monoclonal anti-α-tubulin antibody (1:50,000; Cat. No. T5168, Sigma). Blots were developed using LuminataTM ForteWestern HRP Substrate (Millipore).

Western blots were performed as previously described [36 (link)]. Total cell lysates of cultured cells (60,000 cell/well) were resolved in sodium dodecyl-sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters (Millipore, Billerica, MA, USA) using an Amersham Biosciences semidry Trans-Blot (Buckinghamshire, UK). The membranes were blotted with the following: anti-SMN (1:5000, Cat. No. 610646, BD Biosciences), anti-phospho-Akt (Thr 308) (p-Akt, 1:1000, Cat. No. 9275, Cell Signaling Technology, Danvers, MA, USA), anti-Akt-1 (pan-Akt, 1:10000, Cat. No. SC-1618, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-p44/42 ERK1/2 Thr202/Tyr204 (p-ERK, 1:15000, Cat. No. 9101, Cell Signaling Technology), anti-ERK (pan-ERK, 1:5000, Cat. No. 612641, BD Biosciences), anti-LC3 (1:1000, Cat. No. 2775), anti-phospho-mTOR (Ser2448) (p-mTOR, 1:1000, Cat. No. 5536), anti-mTOR (1:1000, Cat. No. 2972), and anti-p62/SQSTM1 (1:1000, Cat. No. 5114), all from Cell Signaling Technology. Specific protein content per lane was assessed reprobing the membranes with monoclonal anti-α-tubulin antibody (1:50000, Cat. No. T5168, Sigma). Blots were developed using Immobilon® Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).

For immunoblot analysis, anti-SQSTM1, anti-LC3 and anti-BECN1 were purchased from BD Biosciences and Sigma Aldrich, respectively. For immunofluorescence studies, anti-LC3 was purchased from Cell Signaling and anti-SQSTM1 from Abnova. Anti-TLR2 was from Genetex and anti-MyD88 was from Cell Signaling. Monoclonal anti- β-actin was provided by Abcam and a polyclonal anti- GAPDH was from Ambion Life Technologies. Antibodies directed against gD, ICP0, phospho-p38α and histone H3 were from Santa Cruz Biotechnology; the antibody against ICP8 was kindly provided by Pr. Bernard Roizman. Horseradish peroxidase anti-rabbit and anti-mouse antibodies were from Santa Cruz Biotechnology. Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit antibody was from Jackson and Alexa Fluor 350 donkey anti-mouse antibody was from Invitrogen. Spautin-1 (10 μM), 3-methyladenin (10 mM), filipin (2.5 μg/ml) and sucrose (0.45 M) were from Sigma Aldrich.