Express plus

For Western blotting, samples were boiled for 5 min at 95°C for in loading buffer (50 mM Tris pH 6.8, 40% glycerol, 4% SDS, bromophenol blue) without reducing agent before loading onto a 4–16% PAGE gel (GenScript ExpressPlus™). Gels were blotted on PVDF membranes and fixed in 4% formaldehyde for 30 minutes and boiled in PBS for 5 minutes. For dot blotting 50-100ng of protein was blotted on nitrocellulose (Hydrobond-C Extra, GE Healthcare) and these filters were not fixed or boiled. All membranes were blocked in non-fat milk (TBS with 5% non-fat milk, 0.05% Tween 20 and 0.02% sodium azide) for 30 minutes at RT followed by incubation with primary antibodies overnight. membranes were washed in TBS-T (TBS with 0.1% Tween) and incubated with secondary antibodies for 1.5 hour. Membranes were washed, developed using ECL reagent (Pierce™, Thermo Scientific), and subsequently imaged on a LAS-3000 imaging system (Fuji). Blot quantification was done using ImageJ

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.