Gfp rab5

EPEC E2348 strain was kindly provided by Professor Gad Frankel, Imperial College London, UK. Rab5-GFP, Rab7-GFP, and Rab9-GFP, Rab5-DN-mCherry and Rab7-DN-mCherry were from Addgene. VSVG3-SP-GFP was kindly provided by Associate Professor Derek Toomre, Yale University, USA. The plasmid encoding Exo70-GFP was kindly provided by Team Leader Philippe Chavrier, Institut Curie, France. The plasmid encoding TfR-mCherry was kindly provided by Assistant Professor Matthew Kennedy, UC Denver, Colorado, USA. EPEC bacteria expressing GFP were produced by transformation of a GFP plasmid (GFP inserted into the pBluescript SK+ vector, kindly provided by Dr. Stefanie Bechstein, iNANO, AU, DK) into the EPEC E2348 strain and selected with 100 mg/L ampicillin. Before transformation, EPEC bacteria were made competent through a series of resuspensions in 10% glycerol using decreasing volumes after every spin.
Anti-gp135 mAb, clone 3F2/DB [70 (link)], kindly provided by Professor W. James Nelson, Stanford University, California, USA, was used as undiluted hybridoma supernatant. Antibodies against Lipid A were purchased from Abcam.

The pcDNA₃ SPC99_G33L construct was generated using the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies) with pcDNA₃ SPC99 previously described (flammang 2012) and appropriated primers: 5′- AAG GCG CAA TCA TTC TAC TCA TGG TGG GCG GTG - 3′ and 5′- CAC CGC CCA CCA TGA GTA GAA TGA TTG CGC CTT - 3′. The pcDNA₃ SPC99_G29L/G33L plasmid was obtained using the same protocol with the pcDNA₃ SPC99_G33L previously generated and the following primers: 5′- GGG TTC AAA CAA ACT CGC AAT CAT TCT ACT C - 3′ and 5′ - GAG TAG AAT GAT TGC GAG TTT GTT TGA ACC C - 3′). The doxycyclin-inductible pSBtet SPC99 construct used for stable cell line generation was obtained as following. First, the SPC99 fragment was amplified by PCR from the pcDNA3 SPC99 using the following primers (5′– ATA TTA GGC CTC TGA GGC CCC ACC ATG CTG CCC GGT TTG GCA C – 3′ and 5′– GAT GGC CTG ACA GGC CCT AGT TCT GCA TCT GCT CAA AGA ACT TG TAG GTT – 3′) to introduce the SfiI restriction site at both 5′ and 3′ end of fragment. The resulting product was then digested by SfiI and subcloned into the pSBtet vector. All constructs were verified by sequencing. Rab5-GFP, Rab7-GFP and Lamp1-GFP were from Addgene and the SorLA_myc construct was a kind gift from Peter St-George-Hyslop.