Supersignal west pico substrate

Cell extracts were prepared as described previously (Pidoux et al., 2014 (link)). Protein samples were resolved by SDS-PAGE and immunoblotted with antibodies (catalog numbers and supplier are indicated unless stated above) against PKA RIα (0.25 μg/ml; 4D7; BD Biosciences), PKA RIIα (0.25 μg/ml; 612243; BD Biosciences), PKA RIIβ (0.25 μg/ml; 610626; BD Biosciences); PKA Cα/β (0.25 μg/ml; 610980; BD Biosciences); AKAP18 (0.5 μg/ml; WH0009465M1; Sigma–Aldrich); GAPDH (1 μg/ml; G8795; Sigma–Aldrich). After incubation with the appropriate HRP-conjugated secondary antibody, blots were developed by using Supersignal West Pico substrate (Thermo Scientific, Illkirch, France).

Immunoblot analysis was performed as previously described^{29 (link)}. Cells were lysed with RIPA buffer (Beyotime). Protein samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Primary antibodies used include 1/1000 anti-Actin (AF5001), 1/1000 anti-GST (AF2299), 1/1000 anti-Flag (AF0036) (Beyotime), 1/1000 anti-HA (SAB2702196), 1/1000 anti-Myc (SAB2702192), 1/500 anti-SOCS7 (SAB2103430), 1/500 anti-IKKγ (SAB3500414) (Sigma-Aldrich), 1/500 anti-iNOS (2982), 1/200 anti-phospho-IκBα (Ser32/36) (9246), 1/500 anti-IκBα (4814), 1/200 anti-phospho-IRF-3 (Ser386) (37829), 1/500 anti-IRF-3 (11904), 1/200 anti-phospho-STAT2 (Tyr690) (4441), 1/500 anti-STAT2 (4594), 1/500 anti-NF-κB p65 (8242), 1/500 anti-IKKα (2682), 1/500 anti-IKKβ (2678), 1/500 anti-phospho-IKKα/β (Ser176/180) (2697) (Cell Signaling Technology, Inc., Danvers, MA, USA), 1/500 anti-Ubiquitin (sc-271289), 1/200 anti-PTPN14 (Pez) (sc-373766) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1/500 anti-STAT1 (AHO0832), and 1/200 anti-phospho STAT1 (Tyr701) (44–376G) (Invitrogen). Immunoblots were revealed using the SuperSignal west pico substrate (ThermoFisher Scientific, San Jose, CA, USA). For coimmunoprecipitation (Co-IP), the lysates were incubated with appropriate antibodies and Protein A/G beads (ThermoFisher Scientific) overnight at 4 °C followed by immunoblot analysis.

Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 μl of 2X Laemmli buffer. Protein samples were loaded into a 4–20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot^® Turbo™ Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; ref. ^{60 (link)}) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4 °C. The membranes were then incubated with HRP-conjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 h at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal™ West Femto or SuperSignal™ West Pico substrate (Thermo Scientific™) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.

Recombinant proteins were fractionated by SDS-PAGE using a 12% (w/v) polyacrylamide gel. Immunoblot analysis was performed using an anti-His₆ antibody and chemoluminescent detection with SuperSignal West Pico substrate (Thermo Scientific). Blots were developed on Kodak XAR film.

Cells were extracted in RIPA buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with EDTA-free protease inhibitor cocktail (Thermo Scientific) and 1 mM sodium orthovanadate. Protein load was normalized using bicinchoninic acid assay. For SDS-PAGE, 30 μg of cellular protein was loaded per well. Proteins were electrotransferred to PVDF membranes and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody (Jackson Immuno-Research). Bands were imaged using Super Signal West Pico substrate or Femto substrate (Thermo Fisher Scientific).

Cell homogenates were prepared in ice-cold NP-40 lysis buffer [50 mM Tris-HCl, pH 7.4, 10% (v/v) Glycerol; 100 mM NaCl, 2 mM MgCl₂, 1% (v/v) NP-40, freshly added: 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml Pefabloc], boiled in SDS loading buffer and separated by SDS-PAGE. Proteins were transferred overnight on to PVDF membrane (03010040001, Roche, Sigma-Aldrich). The membrane was blocked in 5% non-fat dry milk in TBS with 0.05% Tween 20 (TBST) for 1 h at room temperature (RT) and incubated with primary antibodies overnight at 4°C. The membrane was washed three times with TBST (each wash 5 min) and incubated with the corresponding HRP-conjugated secondary antibody for 1 h at RT. After three washes with TBST the protein bands were detected with Super Signal West Pico Substrate (34078, Thermo Fisher Scientific) according to the manufacturer's protocol, using a ChemiDoc MP Imaging System (Bio-Rad).

HepG2 cells were harvested and boiled with a sample buffer (1 × DB). Proteins were separated by SDS-PAGE and transferred to Poly Vinylidene Di-Fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin or 3% skim milk for 1 h, the membranes were incubated with a primary antibody overnight at 4 °C. After being washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20, the membranes were reacted with an antimouse or rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000) for 1 h at room temperature. Membranes were washed with PBS three times and subjected to immunoblotting using SuperSignal West Pico Substrate (Thermo Scientific, Waltham, MA, USA) or ImmunoStar LD (Wako). Band intensities were quantitatively analyzed using ImageJ. The following antibodies were used: actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), AMPK (#5832, Cell Signaling Technology, Danvers, MA, USA), p-AMPK (#2535, Cell Signaling Technology), ACC (#3662, Cell Signaling Technology), p-ACC (#11818, Cell Signaling Technology), p53 (sc-393031, Santa Cruz Biotechnology), p-p53 (#9284, Cell Signaling Technology), p70S6K1 (#2708, Cell Signaling Technology), p-p70S6K1 (#9234, Cell Signaling Technology), cleaved caspase-3 (#9664, Cell Signaling Technology), caspase-3 (#9665, Cell Signaling Technology).