Quantifying Protein Expression by Western Blot

Protein levels of GFP, dnRhoA, and β-Actin were assessed using Western Blot, following previously described protocols.^{27 (link)} Briefly, 5 μg of protein were separated on pre-cast SDS-PAGE gels (Bio-Rad) and transferred onto Immobilon IP membranes (Millipore) before probing for GFP (1:2500, Abcam, #ab6556), RhoA (1:500, #MA1-134, Thermo Fisher Scientific), β-Actin (1:2500, #ab170325, Abcam), and GAPDH (1:50,000, #PA1-9046, Thermo Fisher Scientific). Primary antibodies were detected using IRDye 800CW goat anti-mouse (#925-332210), IRDye 680RD donkey anti-rabbit (#925-68073), or IRDye 800CW donkey anti-goat (#926-32214) secondary antibodies (1:15,000, LI-COR). Blots were visualized on an Odyssey Infrared Imaging System (LI-COR). Blots were stripped with NewBlot PVDF Stripping Buffer (LI-COR) and reprobed once to allow for detection of all four proteins on the same blot.