Bradford lowry method

HEK293 cells were transfected with miRNA expression cassettes as indicated. At 48 h cells were rinsed once with iced-cold PBS and lysed with Passive lysis buffer (PBL, Promega). Protein concentration was determined by the Bradford–Lowry method (BioRad) and 10 μg of protein loaded on a NuPAGE 3–8% Tris-Acetate gel (Novex Life Technologies). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with a mouse anti-Htt (1:5000, Millipore, CA, USA), or rabbit anti-Beta-actin (1:40000, Sigma) antibodies followed by horseradish peroxidase-coupled antibodies (1:10,000, mouse; or 1:50,000, Rabbit; Jackson ImmunoResearch, West Grove, PA, USA). Blots were developed with ECL-Plus reagents (Amersham Pharmacia). Silencing efficacy was determined by densitometry (n = 4 independent experiments) of protein levels relative to beta actin with the VersaDoc^TM Imaging System (Biorad) and Quantity One^R analysis software.

Snap‐frozen kidneys were ground in liquid nitrogen by a mortar and pestle and then homogenized in lysis buffer (1X RIPA buffer) containing protease and phosphatase inhibitors. Total protein concentrations were then determined using (DCTM Protein Assay II kit) Bradford/Lowry method (Bio‐Rad, Hercules). Protein samples (30 μg) were loaded into the wells of 12% SDS‐PAGE gel. Gels were then transferred to PVDF membranes at 4°C at 30–40 volts overnight. The membrane was then blocked in 5% fat‐free milk, prepared in 0.1% Tween PBS, for 1 hr at RT. Proteins were detected by primary monoclonal antibodies against interleukin 10 (IL‐10), interleukin 4 (IL‐4), interleukin 13 (IL‐13), interleukin 1 beta (IL‐1β), transforming growth factor beta (TGF‐β), tumor necrosis factor alpha (TNF‐α), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Sigma); the latter was used to ensure equal loading of samples. Immunoblots were then probed with the appropriate secondary antibody (anti‐mouse 1:5,000, anti‐rabbit 1:10,000) for 1 hr at RT. Bands were detected with ECL chemiluminescence kit (Thermo Fisher Scientific). The intensity of bands was then determined by densitometry, using the ImageJ software (https://imagej.nih.gov/ij/).