Any kd acrylamide gels

Unless otherwise noted, cells were lysed directly in 1x SDS loading buffer (10% glycerol, 5% BME, 62.5mM TRIS-HCl pH 6.8, 2% SDS, and BPB), boiled, and sonicated (Qsonica Q500). Samples were run on “Any kD” acrylamide gels (Bio-Rad) and transferred to PVDF membranes using a Trans-Blot SD (Bio-Rad) or a Trans-Blot Turbo (Bio-Rad). Blots were blocked in 5% dry milk/TBS-T for 30 minutes to an hour at RT or overnight at 4C. Primary antibodies were diluted in 5% dry milk/TBS-T and added for 1 to 2 hours at RT or overnight at 4C. Blots were washed four times in TBS-T before addition of HRP-conjugated secondary antibody in 5% milk for thirty minutes. Blots were washed four times in TBS-T prior to detection with either ProSignal Dura (Prometheus) or Clarity ECL (Bio-Rad) substrate and exposure to radiography film or imaging on a C-Digit imager (LiCOR).

A quarter of a kidney was homogenized using a IKA^® tissue homogenizer in 500 μL RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) containing Complete^® protease inhibitors (Roche, Basel, Switerland). Subsequently, a Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed to assess the absolute quantity of protein in each sample. Next 10–40 μg of total protein lysate was loaded per lane on precast Any-kD acrylamide gels (Bio-Rad, Hercules, CA, USA) for gel electrophoresis. Using the Trans-Blot^® Turbo™ Transfer system (Bio-Rad), the protein was transferred to Nitrocellulose (Bio-Rad) membranes for further analysis. Membranes were blocked and antibodies incubated o/n at 4 °C in 5% skim milk powder (Nutricia, Zoetermeer, Netherlands) in PBS. The appropriate HRP-labeled secondary antibodies (Dako, Glostrup, Denmark) were incubated for 1 h at room temperature, followed by extensive washing with PBS. Band-visualization was achieved using SuperSignal West-Dura^® Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) for HRP. Next, exposure on UltraCruz^® Autoradiography Film or KODAK-XAR film and development by a Konica developer followed. Finally, band intensities were quantified using ImageJ 1.x [62 (link)].