This is a knockout-validated antibody summary, based on the publications "Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC", as cited below [1] and "The CUL5 Ubiquitin Ligase Complex Mediates Resistance to CDK9 and MCL1 Inhibitors in Lung Cancer Cells" for western blot (figure 4e) [2]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Immunoblots of indicated proteins in cells treated with DMSO or erlotinib (1 mM) for 6 hr to confirm specific knockout of positively selected hits and on-target inhibition of EGFR pathway by erlotinib treatment. b-Actin was used as a loading control. From [1].

Rabbit monoclonal IgG

Company: Abcam

Antibody: Cullin 5/CUL-5

Catalog number: ab184177

Summary: Rabbit monoclonal IgG against a recombinant fragment within human Cullin 5/CUL-5 (aa 150-300). Reacts with mouse, rat and human. Suitable for western blot.

Western blot

Control and CUL-5-KO HCC827 cells. Cells were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific #89901) supplemented with 100 x Protease Inhibitor Cocktail (Sigma #P8340), 100 x Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific #78427) and 25 units/ml Benzonase Nuclease (Sigma #E8263).

5% Blotting-Grade Blocker (Bio-Rad #1706404) in Tris Buffered Saline with Tween 20, pH 8.0 (TBST) (Sigma #T9039) for 1 hr at room temperature.

1:1,000 dilution in 5% BSA (Akron Biotechnology #AK8917-0100) overnight at 4°C.

Horseradish peroxidase (HRP) conjugated secondary antibody diluted in 5% Blotting-Grade Blocker.

Amersham ECL Western Blotting Detection Reagents (GE Healthcare #RPN2106) or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific #34580) and Amersham Hyperfilm ECL (GE Healthcare #28906839).