Expressing Pore-Forming Toxin in E. coli

Plasmid allowing the expression of PFO in E. coli was kindly offered by Daniel Portnoy (DP-4167) [42 (link)]. Plasmid GFPNMHCIIA was a gift from Robert Adelstein through Addgene (#11347) [43 (link)], plasmid tdTomato-F-Tractin [44 (link)] was kindly offered by John Hammer (NIH, Bethesda, MD, USA), and plasmid pUBC eGFP-KDEL was a gift from François-Xavier Campbell-Valois [45 (link)]. The following antibodies were used at 1/200 for immunofluorescence microscopy (IF): rabbit anti-NMHCIIA (Sigma, St Louis, MO, USA), mouse anti-NMHCIIA (Abcam, Cambridge, UK), and mouse anti-KDEL (Abcam, Cambridge, UK). Secondary antibodies were used at 1/200: goat anti-rabbit Alexa Fluor 488 (Invitrogen, Waltham, MA, USA) and goat anti-mouse Cy3 (Jackson ImmunoResearch, West Grove, PA, USA). For IF, F-actin was labeled with rhodamine phalloidin (Invitrogen, Waltham, MA, USA) and the PM with FITC-conjugated WGA (Sigma, St Louis, MO, USA) that recognizes sialic acid and N-acetylglucosaminyl sugar residues at the PM.