Uvp imaging system

Measurement of IL-Ra mRNA expression in Lv.IL-1Ra.copGFP/mBMSCs was performed using RT-qPCR. RNA was extracted from 5×10⁵ Lv.IL-1Ra. copGFP/mBMSCs from P3, P6 and P9 using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. The murine cDNA was synthesized by reverse transcriptase M-MLV (Takara Bio Inc.). The primers used for the amplification are presented in Table I. RT-qPCR was performed by TP800 using SYBR Premix Ex Taq (Takara Bio Inc.), according to the manufacturer's instructions. β-actin was used as a reference. The real-time PCR conditions were as follows: denaturation at 95°C for 10 sec, 40 cycles at 56°C for 20 sec and 72°C for 20 sec. Dissociation was performed for a melting curve analysis to monitor and avoid non-specific amplification as well as primer dimers. The amplified PCR products were separated on a 1.5% agarose gel using electrophoresis (Takara Bio, Inc.), the bands were visualized using ethidium bromide and images were captured with a UVP imaging system (UVP, Upland, CA, USA). The quantitated mRNA values were normalized against the quantities of HPRT mRNA, and results were administered as -fold induction. PCR analysis was performed at P5.

After treatment with WGA or inhibitors, cells were washed twice with 1 x PBS and harvested using RIPA cell lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and 1 x protease inhibitor cocktail. Equal amounts of protein were subjected to 10%–15% SDS-PAGE and electrotransferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked in 5% fat-free milk in Tris-buffered saline with 0.1% Tween 20 to prevent non-specific binding and incubated at room temperature for 1 h. The membranes were incubated with primary antibody at 4°C for 16 h and then incubated with the appropriate secondary antibodies at room temperature for 1 h. Protein signals were detected by chemiluminescence using the horseradish peroxidase substrate Luminol (Millipore) and visualized with a UVP imaging system (UVP, Upland, CA, USA).

Cells or kidney tissues were lysed in RIPA buffer in the presence of 1% PMSF and 1% protease inhibitor cocktail, sonicated and stored at –80 °C. Protein concentrations were measured by BCA assay. Whole-cell lysates were loaded into the wells of 10% SDS-PAGE gel with equal amounts and subjected to electrophoresis, and then transferred to PVDF membrane (Millipore, USA) and blocked in 5% skim milk for 1 h at room temperature. Subsequently, the membranes incubated with indicated primary antibodies and following secondary antibodies. UVP imaging system (UVP, USA) was used to scan the membranes and Quantity One analysis software was applied to quantify the band intensity of blotted proteins.

Frozen muscle tissue was freeze-dried, homogenized, separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and electroblotted onto PVDF membranes as previously described (Rahbek et al., 2015 (link)). Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and used as follows; p-FoxO3a (Ser²⁵³) (conc. 1:1000, cat # 3938) p-ULK1Ser555 (1:2,000; cat. no. 5869) and LC3B (1:1,000; cat. no. 3868). P-FoxO3a was diluted in 1% BSA. The other primary antibodies were diluted in 5% BSA. After overnight incubation in primary antibodies, membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. 6721 ABCAM, Cambridge, UK) in a 1:5,000 solution with 1% BSA. Proteins were visualized by chemiluminescence (Thermo Scientific, Waltham, MA, USA) and quantified with an UVP imaging system (UVP, Upland, CA, USA). Arbitrary protein intensity was normalized to total amount of protein loaded in the corresponding lanes using Stain Free Technology as previously described (Gilda and Gomes, 2013 (link); Gurtler et al., 2013 (link)).