Fig 1. Baculovirus-expressing EGFR production.

A, Map of the EGFR baculovirus using the flashBACULTRA system with a FLAG epitope placed between the leader sequence peptide (Lrig1 SS) and the full-length mammalian EGFR sequence (EGFR). B, Sf9 insect cells were infected with a MOI of 10 of control baculovirus (C) or EGFR baculovirus (E) for 48 hours followed by collection of total cell lysate and Western Blotting to detect EGFR expression. A set of these cells was treated with 10 ng/ml EGF in order to detect ligand-induced receptor phosphorylation. C, Sf9 insect cells were seeded on glass coverslips and infected with an MOI of 10 of control (C) or EGFR (E) baculovirus for 48 hours. Cells were fixed in 4% paraformaldehyde followed by immunostaining with anti-EGFR antibody (in red) with (+P) or without (-P) permeabilization in 100% methanol. The brightfield image shows cell morphology. Cells incubated with secondary antibody in the absence of EGFR antibody (no primary) were used as a negative control. D, FLAG-EGFR is readily purified from Sf9 insect cells as shown by a representative image of anti-EGFR immunoblot of FLAG EGFR purification and E, a Coomassie-stained SDS-PAGE gel. Bovine serum albumin (BSA) was loaded as a protein of known concentration. Total insect cell lysate starting material (SM), purification washes 1, 3 (W1, W3), and elution (E) were loaded to show efficiency of the FLAG-purification.