Phosphoblocker blocking reagent

Cells were lysed in cell lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Sodium Orthovanadate, 10 μM β-glycerophosphate, 5 μM Sodium Pyrophosphate and 0.5% Triton X-100) freshly supplemented with Protease Inhibitor Cocktail (Sigma, Cat. no. P8340) at 4°C. Proteins were purified by centrifugation for 5 min at 7,000 x g, separated on 8% SDS-PAGE and transferred onto the PVDF membrane (Immobilon - P, Merck Millipore, Cat. no. IPVH00010). Membrane was blocked with 5% PhosphoBlocker Blocking Reagent (Cell Biolabs, Cat. no. AKR-104) and probed with antibodies listed in Sup.Table.2. Bands were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Cat. no. RPN2232) on ChemiDoc XRS (Bio-Rad).

Cells were directly lysed with the CelLyticM cell lysis reagent (C2978; Sigma-Aldrich, St. Louis, MO, USA), containing protease inhibitors (04693159001; Roche, Mannheim, Germany) and phosphatase inhibitors (39050; SERVA, Heidelberg, Germany). Whole-cell lysates were passed through a 25-gauge needle ten times before centrifugation. Total protein concentrations were measured using Pierce 660 nm Protein Assay Reagent (22660; ThermoFisher Scientific, Middletown, VA, USA). Whole-cell lysates mixed with Pierce Lane Marker Reducing Sample Buffer (39000; ThermoFisher Scientific) were boiled at 95 °C for 5 min, loaded into each lane of an SDS polyacrylamide gel (456-9034; Bio-Rad), followed by electrophoresis and transferred to a nitrocellulose membrane (10600012; GE Healthcare, Chicago, IL, USA). After blocking with 5% skim milk (190-12865; FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), or the PhosphoBlocker Blocking Reagent (AKR-103, Cell Biolabs, San Diego, CA, USA) for the detection of phospho-proteins, membranes were incubated with primary antibodies at 4 °C overnight. Protein bands were marked by horseradish peroxidase (HRP)-conjugated antibodies and visualized by ECL Prime Western Blotting Detection Reagent (RPN2236; GE Healthcare) and ImageQuant LAS 4000 (GE Healthcare).

Whole-cell protein extracts were obtained by lysing cells with Triton buffer (20 mM Tris-HCL pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with protease and phosphatase inhibitors (10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM phenylmethanesulfonyl fluoride, 5 mM NaF, and 2 mM Na₃VO₄; Sigma). Proteins were quantified by using Bio-Rad Protein Assay (Bio-Rad) and separated in 10-15% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). Membranes were blocked with 2.5% phosphoBlocker Blocking Reagent (Cell Biolabs) in Tris-Buffered Saline (TBS)-Tween 20, and probed with antibodies against: Puma (Abcam), Bim, Noxa (Enzo life sciences), phosphorylated-Ser79 AAC (p-ACC), ACC, phosphorylated-Ser239 VASP (p-VASP), VASP, phosphorylated-Ser235/236 S6rp (p-S6rp), S6rp, phosphorylated-Ser 209 eIF4E (p-eIF4E), eIF4E (Cell Signalling Technology), Mcl-1, Bcl-2 and Bcl-X_L (Santa Cruz Biotechnology). Antibody binding was detected using secondary peroxidase-labeled anti-mouse (Sigma) and anti-rabbit (Cell Signaling Technology) antibodies and chemiluminiscence was detected using a mini-LAS4000 Fujifilm device (Fujifilm). Equal protein loading was confirmed by probing membranes with α-tubulin antibody (Sigma). Densitometric quantification was done by using Image Gauge software (Fujifilm).

Retinas or optic nerves were dissected and ultra-sonicated in ice cold RIPA buffer to obtain total protein extracts as previously described32 (link)33 (link). Western-blot analyses were performed using antibodies raised against PGC1α (Abcam, 1:1000), SDHβ (Abcam, 1:1000), SIRT1 (Abcam, 1:1000), phosphor-AKT Ser 473 (Millipore, Billerica, MA, 1:1000), pan-AKT (Cell Signaling, Danvers, MA, 1:1000) and phosphor-PDK1 (Cell Signaling, 1:1000). β-actin (Sigma, St. Louis, MO, 1:5000) was used as control to normalize protein levels. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis, with 20 μg of protein per lane, and then transferred to nitrocellulose High bound ECL membranes (GE Healthcare Biosciences, Pittsburgh, PA). The membrane was blocked with 5% Phospho blocker Blocking reagent (Cell Biolabs, San Diego CA) or 3% non-fat milk (Bio-rad, Hercules, CA) for 1 hr at room temperature and probed with primary antibody in blocking buffer overnight at 4 °C. After being washed three times using PBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:3000 for 1 hr at room temperature. After three further washes, immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Thermo Scientific, Southfield, MI).

Samples were lysed using cold 1X RIPA buffer (EMD Millipore; 20-188) containing 1X phosphatase and protease inhibitor cocktail tablets (Roche) and a dispersion-based homogenizer (VWR VDI 12). After incubating on ice for 30 minutes, samples were briefly sonicated (Sonics Vibra-cell) and spun at 1,000xg for 10 minutes at 4°C to separate the lipid layer. The supernatant was collected and spun again at 12,000xg for 10 minutes at 4°C and supernatant from this spin was collected and stored at -80°C. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific; 23227). Samples were diluted in water and 4X Laemmli Sample Buffer (Bio-Rad; 1610747) and run on 4-15% SDS-PAGE Mini-PROTEAN TGX gels (Bio-Rad) then transferred to Immobilon-P PVDF membranes (EMD Millipore). Membranes were blocked with 5% PhosphoBLOCKER™ Blocking Reagent (Cell Biolabs, Inc; AKR-103) in TBST for LAMA4 probing, or 5% Non-fat dry milk (LabScientific; M0841) in TBST for other antibodies for 1 hr. Blots were incubated overnight at 4°C in 1% blocking solution with LAMA4 antibody (Invitrogen; mouse mAb, MA5-24650) or β-Actin (CST; Rabbit mAb, 4970). Membranes were then incubated with IRDye secondary antibodies (LI-COR) for 1 hr. Immunodetection was performed using near-infrared Odyssey CLx System (LI-COR). Analysis was performed issuing the Image Studio Software (LI-COR).