Semi dry electroblotting

Northern blotting was carried out according to Ref. [12] (link) with some modifications. Briefly, 20 μg RNA were resolved on denaturing polyacrylamide gels in 1X TBE, transferred onto a positively charged nylon membrane (Roche Diagnostics) using semi-dry electroblotting (Bio-Rad), and immobilized by UV irradiation (Stratagene Crosslinker 120 mJ/cm²). The membrane was pre-hybridized with hybridization buffer at 37 °C for 1 h, and then hybridized overnight at 37 °C with specific oligodeoxynucleotides. The probes were labeled with Digoxigenin using the DIG oligonucleotide 3′END labeling kit (Roche) and detected using the DIG Nucleic Acid Detection Kit (Roche). The following probes were used: RNU44: 5′-AGTTAGAGCTAATTAAGACCT-3′; tRNA-Ile, 5′-UGGUGGCCCGUACGGGGAUCGA-3′; U11: 5′-TCTTGATGTCGATTCCGCACGCAGAGCAATCGAGTTGCCC-3′; hY1: 5′-AAGGGGGGAAAGAGTAGAACA-3′. The probes were synthesized at the Johns Hopkins Genetic Resources Core Facility. The miRCURY locked nucleic acid (LNA) Digoxigenin-labeled probes for detection of miR-19b, miR-21, miR-23a, miR-24, miR-30c, miR-31, miR-99a and miR-191 were purchased from Exiqon.

SDS-page gels were loaded with 25
or 50 μg of total protein and transferred onto a PVDF membrane
(Bio-Rad, 1704274) using semi-dry electroblotting (Bio-Rad, 1704150)
according to manufacturer’s instructions. Mouse primary α-FLAG
antibody (Sigma-Aldrich, F1804) was diluted at 1:1000 in Tris-Buffered
Saline with 1% Casein (Bio-Rad, 1610782) and secondary α-mouse
HRP-conjugated antibody (Cell Signaling, 7076) was used at a 1:3000
dilution in Tris-Buffered Saline with 1% Casein (Bio-Rad, 1610782).
Membranes were incubated in an enhanced chemiluminescence substrate
(ECL, Bio-Rad, 1705062). Tubulin was detected with a human α-Tubulin
Rhodamine-conjugated antibody (Bio-Rad, 12004165) at a 1:3000 dilution
in Tris Buffered Saline With 1% Casein (Bio-Rad, 1610782).

Pre-treated myotubes, with or without insulin-stimulation (100 nM for 10 min), were lysed in NP40-lysis buffer (10 mM Tris, 150 mM NaCl, 7 mM EDTA and 0.5% NP-40) supplemented with protease and phosphatase inhibitors, sonicated and spun at 14,000 g for 15 min at +4°C. Protein concentration was determined with BCA Protein Assay kit. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membrane by semi-dry electroblotting (Bio-Rad) and blocked with 5% (w/v) milk – 0.1% (v/v) Tween-TBS (TBST). Phosphorylated proteins were detected from membranes incubated with pAkt-Ser⁴⁷³, pAkt-Thr^308, pAS160-Thr⁶⁴², pGSK3β-Ser⁹ and pAMPK-Thr¹⁷² primary antibodies at a dilution of 1:1000 in 5% (w/v) BSA-TBST overnight at +4°C. Primary antibodies were probed with HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence. Total target proteins were detected with corresponding antibodies after treating the membranes with stripping buffer (62.5 mM Tris pH 6.8, 2% (w/v) SDS, 0.7% (v/v) β-mercaptoethanol) for 45 min in +45°C shaker. Intensities of the proteins of interest were quantified using Fiji software (30 (link)) or Image Lab 5.1 software (Bio-Rad). Values of the intensities of the phosphorylated proteins were normalized to the intensity of their corresponding total protein. Data were normalized to the insulin-stimulated control sample of each subject.