Mouse anti beta actin antibody

Sirt1 activity was evaluated from cytoplasmic compartment using a fluorometric assay (SIRT1 fluorimetric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Expression of Sirt1 protein was assessed using western blot as previously described [18 (link)]. Briefly, 10 microgram of cellular extracts were resolved on 10% SDS-PAGE using a Mini-PROTEAN 3 Cell (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were electrotransferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Saclay, France) using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The membranes were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary immunoglobulin raised against the appropriate species; bands were detected using the ECL Plus kit (Amersham Biosciences). The primary antibodies used for western blot are as follows: rabbit anti-Sirt1 antibody (1:1000 dilution; Cell Signaling Technology, Beverly, MA) and mouse anti-beta-actin antibody (1:5000 dilution; Sigma-Aldrich, Saint-Louis, MO). Horseradish peroxidase-conjugated secondary antibodies goat anti-rabbit (1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-mouse (1:5000 dilution; DakoCytomation, Trappes, France) were used.

Western blot analysis was performed on fully-confluent Cre-responsive cell lines harvested from 24-well plates 6 or 22 hours after the single administration of PTD-Cre (15 nM) or syn-mRNA-Cre (2.1 nM). The cells were lysed using RIPA buffer composed of 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0 [27 (link)]. Cell lysates (14 μg total protein per well) were mixed with 4x Laemmli loading buffer containing 8% SDS, 40% glycerol, 0.02% bromophenol blue, 250 mM Tris, and 20% 2-mercaptoethanol (all from Sigma-Aldrich), pH 6.8, heated at 95°C for 3 min, and run on a 15% polyacrylamide gel and transferred to PVDF membranes (Merck Millipore) using a Pierce G2 electroblotter (Thermo Fisher Scientific). The membranes were blocked with 3% BSA (Sigma-Aldrich). Primary antibodies included a rabbit anti-T7 antibody (Abcam, Cambridge, UK) for detecting Cre recombinase (1:2000 dilution) and a mouse anti-beta-actin antibody (Sigma-Aldrich) as a loading control (1:7500 dilution). The secondary antibodies included goat anti-rabbit IgG-HRP (Merck Millipore) and rabbit anti-Mouse IgG-HRP (Thermo Fisher Scientific), each diluted 1:50000. Chemiluminescent SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used for detection. The signals were acquired using a G:BOX Chemi XR5 (Syngene, Cambridge, UK).

Biotin-conjugated goat anti-HA IgG was from Vector laboratories (Burlingame, CA). Streptavidin-HRP, Alexa-488 conjugated streptavidin, and rhodamine-conjugated streptavidin were from Jackson ImmunoResearch Laboratories (West Grove, PA). DMEM, mouse anti-beta-actin antibody, sodium bicarbonate and p-nitrophenyl acetate (NPA) were from Sigma-Aldrich (St. Louis, MO). 4',6-diamidino-2-phenylindole (DAPI) and RPMI-1640 were from Invitrogen (Carlsbad, CA). Bovine calf serum (BCS) was from HyClone (Logan, Utah). ³H-thymidine was from PerkinElmer (Boston, MA).

The mouse retinas were homogenized, the extracted protein concentration was calculated, the retinal proteins were separated with SDS-PAGE, and the retinal proteins were transferred to a PVDF membrane, according to a method previously described^{65 (link)}. The membrane was blocked with 1% skim milk in Tw-PBS for 1 h at room temperature and then incubated with rabbit anti-RBPMS antibody (Abcam; dilution 1:1000) overnight at 4 °C. After washing the membranes with Tw-PBS, they were incubated with HRP- conjugated donkey anti-rabbit IgG (Sigma; dilution 1:5000) at room temperature for 1 h. The immunoreactive signal was developed with ECL prime reagent (GE Healthcare, Piscataway, NJ) and was captured with ChemiDoc (Bio-Rad). The membranes were then reblotted with Restore Western Blot Stripping Buffer (Thermo Scientific) and incubated with mouse anti-beta-actin antibody (Sigma; dilution 1:5000) as an internal control.

HEK293T cells were lysed at 4 °C in a solution of 150 mM NaCl, 5 mM EGTA, 50 mM Tris, pH adjusted to 7.5 with NaOH, Triton X-100 0.5 % (v/v), and protease inhibitors (1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin). After centrifugation at 12,000×g for 10 min, 5× Laemmli buffer containing 100 nM dithiothreitol was added to the supernatant. Lysates were subjected to SDS–PAGE 8 % (w/v) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were incubated with 5 % (w/v) non-fat dried milk in TBS-T (137 mM NaCl, 0.2 % (v/v), Tween-20, and 20 mM Tris/HCl, pH 7.6). Immunoblots were incubated overnight at 4 °C with either a mouse anti-beta-actin antibody (1:1,000) (Sigma, MO, USA) or a goat anti-calpain-1 (1:150) (Santacruz, Dallas, TX) diluted in 1 % (w/v) milk in TBS-T. PVDF membranes were incubated 1 h at room temperature with a sheep horseradish peroxidase-conjugated anti-goat (1:2,000) or anti-mouse (1:10,000) antibody (Sigma, MO, USA) in TBS-T. Afterwards, blots were visualized using the enhanced chemiluminescence system (ECL, Thermo Fisher).

Cells were lysed with RIPA buffer (Millipore, Billerica, MA, United States) that contained phosphatase and proteinase inhibitors. The protein concentration of the lysates was measured by the Bradford assay (Bio-Rad, Hercules, CA, United States). Aliquots of lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blot analysis. Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States). Secondary antibodies were a horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (1:10,000, GE Healthcare, Piscataway, NJ, United States) and HRP-linked anti-rabbit IgG antibody (1:10,000, GE Healthcare). Immunoreactive proteins were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, United States) with an ImageQuant LAS4,000 (GE Healthcare).