I am converting a script to use Gtk3 using the migration guide (Porting GTK2 to GTK3). I converted my import pygtk to a from gi.repository import Gtk a
Well, the solution is pretty obvious, after calling to Gtk.Builder() one needs to convert the old glade interface with the gtk-builder-convert command to get the interface file in the right version.
$ gtk-builder-convert myui.glade myui.ui
And then, in the python script:
from gi.repository import Gtk
builder = Gtk.Builder()
builder.add_from_file("myui.ui")
Thanks to Riccardo.