Mouse anti gfp

All chemicals were purchased in pro analysi grade from Merck KGaA (Germany) or Carl Roth GmbH. Antibodies for western blot were rabbit anti-Bok (Abcam; EPR15331), mouse anti-GFP (BioLegend; Clone B34) and rabbit anti-Tom20 (Santa Cruz Biotechnology; FL-145), mouse anti-β-actin (Sigma; clone AC74), anti-Mcl-1 (Santa Cruz Biotechnology, H-260) anti-mouse-HRP and anti-rabbit-HRP (Jackson Laboratories).

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.

7.5 μg of mouse anti‐GFP (BioLegend, San Diego, CA, USA) or rabbit anti‐mCherry (Thermo Fisher Scientific) antibody were cross‐linked to 50 μL of magnetic Dynabeads protein G (Thermo Fisher Scientific) with BS3 cross‐linking reagent (Thermo Fisher Scientific) as per manufacturer's instruction. The incubation time was extended to 1 h at 4 °C, and the quenching was extended to 30 min. Lysates (500 μg of protein) were applied to the bead–antibody complex and allowed to incubate with rotation at 4 °C overnight. Beads were washed in triplicate with eight beads volumes of 1× TBS containing 0.1% Tween‐20 (TBS/Tween). The beads were resuspended in 100 μL of TBS/Tween and transferred to a fresh microcentrifuge tube to avoid eluting proteins bound to the tube wall. Immunoprecipitated proteins were released from the antibody bead complex by heating in 30 μL 1× SDS loading buffer and supernatant transferred to a fresh microcentrifuge tube.