The actin and microtubule network regulator WHAMM, is identified as a key kidney disease risk gene
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Original blots associated with this original research article.
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Approximately 15 mg of kidney tissue was used to prepare protein sample. Briefly, tissue was homogenized (#LR60902, OMNI-GLH) in SDS-blue loading buffer (#7722, CST) with 42 mM DTT. The proteins were then separated on SDS-PAGE gels and transferred onto a PVDF membrane at 100 V for an hour on ice. Then, the membranes were blocked in 3% non-fat dry milk powder in tris-buffer saline containing 0.1% Tween-20 (TBST) solution for 30 minutes at room temperature. Next, the blots were incubated with primary antibodies overnight at 4°C. After the primary antibody incubation, the blots were washed three times with TBST and probed with IRdye-conjugated or HRP-conjugated secondary antibodies for an hour at room temperature. Finally, the blots were scanned at excitation wavelengths of 600, 700, and 800 or chemiluminescence using a Li-COR imager (Odyssey® Fc) and analyzed using Image Studio software. Blot quantifications were performed in Image J software. Autophagy flux was calculated as described.
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- University of Pennsylvania