Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Cas9 (+), using Cas9 (7A9-3A3) Mouse mAb (upper) and β-Actin (13E5) Rabbit mAb #4970 (lower).
Immunohistochemical analysis of Cas9 expression in Nprl2-deficient 293 (left, positive) and untreated 293 (right, negative) cell pellets using Cas9 (7A9-3A3) Mouse mAb. Nprl2 expression was knocked out in the Nprl2-deficient cells by transient transfection of Cas9 and Nprl2-specific guide sequences. 293/Nprl2 -/- cells were kindly provided by Rachel Wolfson, Lynne Chantranupong, and David Sabatini of MIT, Cambridge, MA.
Confocal immunofluorescent analysis of Cas9 expression in Nprl2-deficient (left) and untreated 293 cells (right) using Cas9 (7A9-3A3) Mouse mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). Nprl2 expression was knocked out in the Nprl2-deficient cells by transient transfection of Cas9 and Nprl2-specific guide sequences. 293/Nprl2 -/- cells were kindly provided by Rachel Wolfson, Lynne Chantranupong, and David Sabatini of MIT, Cambridge, MA.
Flow cytometric analysis of HEK-293 cells untreated (blue) or transfected with NPRL2 CRISPR/Cas9 KO plasmid (green) Cas9 (7A9-3A3) Mouse mAb. Anti-mouse IgG (H+L), F(ab') 2 fragment (Alexa Fluor ® 488 Conjugate) #4408 was used as a secondary antibody.