Polyvinylidene difluoride membrane

Cells were washed with cold phosphate-buffered saline (PBS) and re-suspended in ice-cold radio-immunoprecipitation assay (RIPA) buffer (20 mM Tris- hydrochloric acid in pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate and 1 mM sodium vanadate) containing protease inhibitor mixture. Following cell lysis and centrifugation at 14000 × rpm for 10 min at 4°C, the resulting supernatant was collected as the total cell lysate. Briefly, the lysates containing 30 μg proteins were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis (Life Technologies) and transferred to polyvinylidene difluoride membranes (BIO-RAD laboratories). Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences). All target proteins were immunoblotted with appropriate primary and peroxidase conjugated secondary antibodies. Quimioluminiscence bands were detected with Bio-Rad ChemiDoc MP Imaging System.

SDS-PAGE was carried out on tissue homogenates with each sample standardized to 1.5 μg DNA per well. Proteins were separated on 4%–12% Bis-Tris Plus SDS gels and transferred to 0.45 μm polyvinylidene difluoride membranes (Life Technologies, Carlsbad, USA). Membranes were incubated overnight at 4°C with primary antibodies against carbonic anhydrase IX (CA-IX) (1/200, R&D Systems, AF2188), glucose transporter 1 (GLUT1) (1/1,000; Abcam, ab32551), phosphorylated histone protein γH2AX (1/2000; Abcam, ab11174), or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary horseradish peroxidase-conjugated antibodies (1/5,000, DAKO). β-Actin was used as loading control. For GLUT1, the same positive control (20 µg protein of 1% O₂ treated T24 cell lysate) was run on each gel to normalize signals between the blots. For CA-IX, a clear cell renal cell carcinoma tissue sample (University of Otago Human Ethics committee H14/020 (37 (link)), adjusted to 1.5 μg DNA per well, was similarly run on each gel. For γH2AX, a positive control (WiDr cells from ATCC treated for 4 h with 20 mM ascorbate, 20 µg protein) was run on each gel. Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA), and quantified with the Alliance 4.7 imaging system and the ImageJ software (36 (link), 37 (link)).

Tissues (lungs, SCAT and EWAT) were homogenized in lysis buffer supplemented with protease inhibitors (cOmplete^TM Protease Inhibitor Cocktail, Sigma-Aldrich). Whole tissue protein extracts (40–50 µg) were fractionated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a transfer apparatus as per the manufacturer’s instructions (Life Technologies). Membranes were blocked with 5% bovine serum albumin (BSA) in PBS/0.1% Tween for 1 h, washed and probed overnight at 4 °C with polyclonal antibody against UCP1 (1:500) (Abcam, Cambridge, UK). Membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (KPL, Milford, MA, USA) for 2 h. After three washings, blots were developed with the ECL system (Bio-Rad Laboratories) according to the provided protocol. GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) was used as protein loading control.

Each tissue was homogenized in micro tubes with lysis buffer (CelLytic™ MT cell lysis Reagent: SIGMA-Aldrich, Japan) containing protease inhibitor (SIGMA-Aldrich Japan) and 0.2% SDS. The protein concentration was measured by the Bradford method. A 50 μg aliquot of protein was separated by SDS-PAGE using 4-12% Bis-Tris gel and then transferred onto polyvinylidene difluoride membranes (Life Technology, USA). The membranes were blocked with membrane blocking reagent (GE Healthcare, Buckinghamshire, UK) for 1 hour, and then incubated for 2 hours with rabbit polyclonal primary antibodies against either UCP1 (1:2500; ab1983, Abcam, Cambridge, UK), UCP2 (1:2500; ab97931, Abcam), UCP3 (1:2000; ab3477, Abcam), CPT-2 (1:500; sc-20671, Santa Cruz, Santa Cruz, USA), MCAD (1:1000; sc-98926, Santa Cruz) or α-tubulin (1:2000; ab4074, Abcam). After the primary antibody reaction, the membranes were incubated for 1 hour with the appropriate horseradish peroxidase-conjugated secondary antibody (1:100000). Immunoreactivity was detected by chemiluminescence using the ECL select™ Western Blotting Reagent (GE Healthcare, Buckinghamshire, UK). Fluorescence band images were analyzed using Just TLC (Liponics, Tokyo, Japan) analysis software. Each value was normalized relative to α-tubulin.