Chemidoc it imaging system

Isolated cell protein was recovered after lysis of cells with RIPA lysis buffer (ThermoFisher Scientific, Carlsbad, CA, USA) with protease inhibitor added (Protease inhibitor cocktail I; Calbiochem, Gibbstown, NJ, USA). The homogenate was centrifuged at 15,000× g for 10 min at 4 °C, and total protein concentration of the supernatant was measured (BCA Protein Assay kit, ThermoFisher Scientific). Forty micrograms of total protein were separated by SDS-PAGE on a 4%–12% Tris–HCl gel and dry-transferred onto nitrocellulose membranes (iBlot; Invitrogen Corp.). Membranes were blocked in Tris buffer (pH 7.4) with 5% dry milk powder for 1 h at room temperature, then incubated with a rabbit anti-HCAR2 IgG (PA5-90579 diluted 1:1,000; Invitrogen, ThermoFisher Scientific) for 1 h at room temperature. After washing, membranes were incubated for 1 h at room temperature with a secondary goat anti-rabbit IgG (7074S; Cell Signaling Technology, Beverly, MA, USA) diluted 10,000-fold in Tris buffer (pH 7.4). Immunodetection was performed by chemiluminescence (West-Dura; ThermoFisher Scientific), and band images were visualized using a photo documentation system (ChemiDoc-It Imaging System; Bio-Rad, Hercules, CA, USA).

Northern blot analysis of milRNA identification was performed according to the protocol of Kim et al. with double-labeled digoxigenin (DIG) oligonucleotide probes instead of locked nucleic acids (LNA) probes [51 (link)]. Briefly, total RNA samples (5–15 ug) from the two different developmental stages were resolved on a 15% denaturing polyacrylamide gel with 8M Urea in 1X TBE. The RNA gels were then transferred to Hybond-N+ (Amersham Biosciences) at 10–15 V (30–60 min) using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Cross-linking, hybridization and membrane detection were performed as previously described [51 (link)]. Cross-linking was performed using freshly prepared 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) reagent at 60 °C for 1 hr. Membranes were hybridized overnight in ULTRAhyb^™ hybridization buffer (Ambion) with specific double DIG-labeled oligonucleotide probes synthesized by Integrated DNA Technologies at 37 °C. Sequences of the probes used against the putative milRNAs were as follows: ccin-milR-12c, 5’-AAAGGTAGTGGTATTTCAACGGCGCC-3’; ccin-milR-13e-5p, 5’-AGTCCCTACTAGGTCCCGAG-3’. Probe detection was performed using DIG luminescent detection kit in accordance with the manufacturer’s instructions (Roche) and photoemissions were detected using the ChemiDoc-It Imaging System (Bio-rad).

Thirty-five micrograms of muscle protein from each sample was separated by SDS-PAGE using a 10% gel for the protein kinase B (PKB) and ubiquitin-binding protein (p62) or 15% gel for the microtubule-associated protein light chain 3 (LC3) I/II. The proteins were then transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA) and blocked for 1 h at room temperature (RT) with 5% bovine serum albumin (VWR Life Science, Radnor, PA, USA) diluted in Tris-buffered saline [2.42 g Tris Base (RPI), 8 g NaCl (Thermo Fisher Scientific) and Tween 20 (0.1%; VWR Life Sciences)] (TBST). The membranes were incubated overnight at 4 °C with the primary antibody (dil. 1:1000, Cell Signaling, Danvers, MA, USA), washed the following day 3 × with PBST, and next incubated with the secondary antibody solution (goat anti-rabbit IgG, dil. 1:5000, Invitrogen) for 1 h at RT. An enhanced chemiluminescence kit (Amersham ECL Prime; GE Healthcare, Little Chalfont, UK) was used to develop the membranes, which were imaged on a ChemiDoc-It Imaging System (BIO-RAD, Hercules, CA, USA). Bands were quantified with Image Lab Software (Version 6.0.1; BIO-RAD).