Fig. 2
From: Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection
Identification of knockout lines by protein dot blot. a Schematic of the procedure: after transfection, cells are plated at limiting dilution in 96-well plates, and isolated colonies are picked and expanded. Lysates from clonal populations are blotted on a nitrocellulose membrane and probed for the protein of interest, along with a parallel control protein blot. b One batch of isolated clones (from a total of two) is shown after dot blotting with HuR antibody (left), and separate blotting for a control protein, Pum2 (right). Clones with insufficient control protein signal (<two fold of the membrane background), denoted by an x, likely arose due to pipetting or blotting error, and were excluded from further analysis. Circles denote clones that validated (green) or did not validate (grey) by subsequent western blot. Clone HuR-5.B5 is designated by a star. Boxes around the blots indicate clones isolated from HuR-3, 4, and 5 targeting constructs (from top to bottom respectively)