Methanol

Spot urine was collected and 2 μL of each urine sample, and BSA standards at 0, 1, 3, and 10 μg, were separated by SDS‐PAGE on a NuPAGE Novex 4–12% Bis‐Tris gel (Thermo Fisher Scientific). After electrophoresis, the gel was immersed in fixing solution (50% (v/v) methanol (Fisher Scientific), 10% (v/v) acetic acid (Acros)) for 1 h, incubated for 20 min with agitation in staining solution (0.1% (w/v) Comassie brilliant blue R‐250 (Bio‐Rad), 50% (v/v) methanol, 10% (v/v) acetic acid), and finally incubated several times in destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid) until destained.
Urinary albumin content in spot urine was assessed using Albuwell M indirect ELISA (Exocell) and urinary creatinine content was measured, using Creatinine Companion kit (Exocell). Both measurements were performed according to manufacturer′s instructions. Albumin:creatinine ratio was calculated by dividing μg albumin/mL urine with mg creatinine/mL urine, getting the ratio μg albumin/mg creatinine.

Proteins were resolved by SDS-PAGE as described [31] (link). Gels were transferred to PVDF (polyvinylidene difluoride) membranes pretreated with methanol (Biorad, Hercules, CA) in 25 mM Tris, 192 mM glycine. Membranes were blocked with 1% BSA in PBS containing 0.1% Tween 20 for 1 hour at room temperature. Membranes were then probed with the primary antibodies for either overnight at 4°C or 2 hours at room temperature and incubated with Horseradish peroxidase-conjugated secondary antibodies (Sigma). Signals were enhanced with chemiluminescence reagents (Perkin Elmer Life Sciences, Boston, MA) for detection of Western signals using a Fujifilm LAS-4000 scanner or Autoradiography Film (Molecular Technologies, St. Louis MS).

Lysis of cells was performed with NP40-containing lysis buffer (150 mM NaCl, 50 mM Tris at pH 7.5–7.8, 5 mM NaF, 0.5% NP40, 1 mM sodium vanadate, Complete protease inhibitor cocktail (Roche) for 10 min on ice followed by centrifugation to remove cell debris. For nuclear proteins, NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used according to the manufacturer’s protocol. Protein concentrations were determined with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on precast 4–15% Mini Protean TGX gels at 190 V using the Mini Protean electrophoresis system and TGS buffer and were blotted onto nitrocellulose membranes at 70V for 2 h in a Mini Trans-Blot Cell by using the TG buffer supplemented with 20% methanol (all BioRad). All antibodies were used as recommended by the manufacturer.