Proteins were separated by SDS-PAGE on 4%–20% MOPS-acrylamide gels (GenScript Express Plus, M42012) and electrophoretically transferred onto PVDF membranes. Immunodetection was performed using the iBind Flex Western Device (Thermo Fisher, SLF2000). Antibodies were used at the following concentrations: 1:25,000 mouse anti-Actin (Chemicon/Bioscience Research Reagents, MAB1501), 1:500 mouse anti-ubiquitin (Santa Cruz, sc-8017), 1:2000 mouse anti-HA (BioLegend), 1:1000 mouse anti-mCherry (Invitrogen), and 1:500 mouse anti-GFP (BioLegend).
HRP secondary antibodies were used as follows: 1:500 to 1:1000 anti-mouse (BioRad, 170-6516), 1:500 to 1:1000 anti-rabbit (BioRad, 172-1019). Signal was detected using Pierce ECL Western Blotting Substrate (Thermo Scientific, 32106). Densitometry measurements were performed blind to genotype and condition using Fiji software [49 (link)]. Signal was normalized to Actin [114 (link), 115 (link)]. For comparisons involving a genetic manipulation, the value for the control genotype was then set at 1. Normalized immunoblot data were log2-transformed to stabilize variance, and means were compared using Student t test. Significant results were defined as increases of at least 1.25-fold or decreases of at least 0.8-fold with p < 0.05. Each experiment was performed using at least three independent biological replicates.
HRP secondary antibodies were used as follows: 1:500 to 1:1000 anti-mouse (BioRad, 170-6516), 1:500 to 1:1000 anti-rabbit (BioRad, 172-1019). Signal was detected using Pierce ECL Western Blotting Substrate (Thermo Scientific, 32106). Densitometry measurements were performed blind to genotype and condition using Fiji software [49 (link)]. Signal was normalized to Actin [114 (link), 115 (link)]. For comparisons involving a genetic manipulation, the value for the control genotype was then set at 1. Normalized immunoblot data were log2-transformed to stabilize variance, and means were compared using Student t test. Significant results were defined as increases of at least 1.25-fold or decreases of at least 0.8-fold with p < 0.05. Each experiment was performed using at least three independent biological replicates.