Mouse anti s6

Western blot analysis was performed as described previously [52 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-NOXA, rat anti-BMF (Alexis Biochemicals, Grünberg, Germany), mouse anti-AKT, mouse anti-BCL-2, mouse anti-BAX, rabbit anti-BAK (BD Bioscience), rabbit anti-caspase-3, mouse anti-caspase-9, rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, (Cell Signaling, Beverly, MA), rabbit anti-MCL-1 (Stressgene Bioreagents, Victoria, BC). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA)) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.

The following primary antibodies used in this study were purchased from commercial suppliers: rabbit anti-Akt, p-Akt (S473 and T308), S6, p-S6 (S235/236), p-mTOR (S2448), mTOR, Beclin-1, LC3B, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mouse anti-S6 from Cell Signaling (cat# 4691, 4060, 2965, 2217, 4858 or 2211, 5536, 2983, 3738, 2775, 5174, 2317); rabbit anti-Vps15 (A302-571A) from Bethyl Laboratories; rat anti-perlecan from Millipore (MAB1948P); rat anti-CD11b from Fisher (BD Biosciences, BDB550282); dystrophin (MANDYS16) and embryonic myosin heavy chain (eMHC, F1.652) from the Developmental Studies Hybridoma Bank (DSHB); and rabbit anti-collagen VI (ColVI, 70R-CR009x) from Fitzgerald Industries. Antibodies detecting functionally glycosylated αDG (IIH6) and β-dystroglycan protein (βDG, 7D11) have been described previously [1 (link), 35 (link)] and were a gift from Dr. Kevin Campbell (U. Iowa) or purchased from DSHB. αDG-core antibodies (45-3, 5-2) were reported recently [36 (link)]. Secondary antibodies conjugated to horseradish peroxidase or Alexa Fluor® 488 or 546 were purchased from Millipore, Jackson ImmunoResearch, or Life Technologies.

Cells were lysed using boiling lysis buffer (1% SDS, 10 mM Tris.HCl, pH7.4). Protein concentrations were measured using BCA protein reagent (Thermo Scientific) and equal amounts of protein were separated on 10% or gradient polyacrylamide gels and transferred onto PVDF membranes [69 (link), 81 (link)]. The following antibodies were used: rabbit antibodies against phospho-pS6K(T389), phospho-S6(S235/236) and phospho-S6(S240/244), S6K, phospho-4EBP1(T37/46), phospho-AKT(S473) and phospho-AKT(T308), AKT and mouse anti-S6 – from Cell Signaling Technology (Danvers, MA); mouse monoclonal antibodies against cyclin D1 and rabbit anti-actin were from Santa Cruz Biotechnology (Paso Robles, CA) and Sigma-Aldrich (St. Louis, MO), respectively.

The following primary antibodies were used for Western blotting: rabbit-anti-Insulin receptor β (Santa Cruz, #711), rabbit-anti-phospho Akt (Ser473) (Cell Signaling, #4060), rabbit-anti-Akt (Cell Signaling, #9272), rabbit-anti Gsk3β (Cell Signaling, #9315), rabbit-anti-phospho Gsk3β Ser9 (Cell Signaling, #5558), rabbit-anti FoxO1 (Cell signaling, #2880), rabbit-anti-phospho FoxO1 Ser256 (Cell Signaling, #9461), rabbit-anti p70 S6 kinase (Cell Signaling, #2708), mouse-anti-phospho p70 S6 kinase Thr389 (Cell Signaling, #9206), mouse-anti S6 (Cell Signaling, #2317), rabbit-anti-phospho S6 Ser235/236 (Cell signaling, #4856), rabbit-anti 4E-BP1 (Cell Signaling, #9644), rabbit-anti-phospho 4E-BP Thr37/46 (Cell Signaling, #9459), mouse-anti-Gapdh (Abcam #ab8245), rabbit-anti-phospho HSL Ser660 (Cell Signaling, #4126), rabbit-anti-phospho HSL Ser563 (Cell Signaling, #4139), rabbit-anti-HSL (Cell Signaling, #4107), rabbit-anti-PLIN1 (Cell Signaling, #9349) and rabbit-anti-CD36 (Cell Signaling #14347). Secondary antibodies used for western blotting were goat-anti-rabbit IgG-HRP conjugate (Biorad, #1706515) and goat-anti-mouse IgG-HRP conjugate (Biorad, #1706516).

BEZ235, TSA and 3-MA were purchased from Selleck company (Selleck, Shanghai, CHINA). Antibodies for western blotting and flow cytometry were: mouse anti-cleaved caspase-3, mouse anti-cleaved caspase-8, mouse anti-cleaved caspase-9, mouse anti-p-Akt, mouse anti-Beclin-1, mouse anti-LC3, mouse anti-S6, mouse anti-p-S6, mouse anti-4EBP1, mouse anti-p-4EBP1, mouse anti-mTOR (Cell Signaling, USA), mouse anti-PARP-1 (Abcam, USA).

Brain and spinal cord sections were homogenized in lysis buffer (10 mM Tris, pH 7.4, 2 mM EDTA, 0.25 M sucrose) supplemented with protease (Sigma) and phosphatase inhibitor cocktails (Thermo Scientific). The lysates were incubated on ice for 30 min, vortexed, and centrifuged at 14.000 g for 10 min at 4°C. Protein concentrations were determined using the BCA assay (Thermo Scientific). Equal amounts of protein (i.e., 20 μg) were mixed with Laemmli loading buffer (Bio-Rad) and loaded on a 10% or 18% SDS-PAGE gel. After separation, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore), which were incubated with the following primary antibodies overnight: mouse anti-p62/SQSTM1 (1:1000, Abcam), rabbit anti-LC3B (1:1000, Novus biologicals), mouse anti-S6 (1:500, Cell Signaling), rabbit anti-phospho-S6 (1:500, Cell Signaling), mouse anti- β-tubulin (1:1000, Abcam), and rabbit anti-β-actin (1:1000, Abcam). Thereafter, membranes were washed and incubated with the following secondary antibodies for 1 h at RT: anti-rabbit IRDye800CW and anti-mouse IRDye680RD (LI-COR Biosciences). Signals were detected using the Odyssey Imaging System (LI-COR Biosciences) and densitometric values were determined and quantified at non-saturating exposures using the ImageJ software (Schneider et al. 2012 (link)), and normalized against the loading controls.