Revert 520 total protein stain

Whole cell lysate was collected from Human Brain Endothelial Cells-5i (HBECs, American Type Culture Collection [ATCC]) grown in 24-well tissue culture dishes in 2x Laemmli Sample Buffer. Samples were incubated for 5 min at 95°C to denature proteins and then loaded onto 4%-20% gradient SDS-PAGE gels (BioRad). Proteins were transferred to nitrocellulose blotted with RhoA-GTP (New East Biosciences), RhoA (Pan) ROCK, phosphor-MLC, and MLC (Cell Signaling Technologies) mAbs diluted (1:1000) in 1x TBST (1% BSA). WBs were then incubated with GAM 680 and GA-rabbit 800 (LiCOR Biosciences) secondary Abs. Revert 520 Total protein stain (LiCOR) was used for WB normalization. Blot images were collected using an Odyssey FC imager (LiCOR) and protein levels were quantified using LiCOR Image Studio software.

3 μl cell lysate containing 8 μg protein was spotted onto nitrocellulose membrane for each sample in triplicate. The membrane was allowed to dry for 15 min prior to staining with Revert 520 Total Protein Stain (Li-Cor Cat. # 926–10010) according to the manufacturer’s protocol (https://www.licor.com/documents/1o8anlg26tnwqkj135ki6bo61fy4ztmi). The membrane was imaged on the Li-Cor Odyssey M imaging system using the 520 channel to obtain a total protein quantification for normalization. The membrane was then blocked with Odyssey Blocking Buffer, incubated with rabbit anti-HUWE1, washed with TBST, incubated with IRDye 680RD donkey anti-rabbit IgG secondary antibody, washed with TBST followed by TBS, and imaged using the Li-Cor Odyssey M imaging system. Acquisition parameters in the manufacturer’s Li-Cor Odyssey Image Studio Lite software were set so as to avoid saturated pixels in the dots of interest, and dots were quantified using background subtraction. The integrated intensity for HUWE1 was normalized to that for Revert 520 Total Protein Stain in the same blot, and the average ± SD from triplicate dot blots was used to represent the data.