Metamorph Imaging System software was used to run the microscope and obtain the images (Universal Imaging Corp., Pennsylvanian). Immunolocalization reagents Primary antibodies consisted of a mouse monoclonal anti-β-Spectrin II (used at 2.5 μg/ml for immunofluorescence, 0.025 μg/ml for westerns) (Becton Dickinson), a rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.02 μg/ml for westerns) (Santa Cruz Biotechnology), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence

and 0.017 μg/ml for westerns)(Sigma Aldrich), and rabbit anti-calnexin (Becton Dickinson) (used at 1:2000). Secondary antibodies included goat anti-mouse or anti-rabbit selleck screening library antibodies conjugated to AlexaFluor 568 or 594 (used at 0.02 μg/ml) (Invitrogen). For F-actin staining

AlexaFluor 488 conjugated phalloidin (Invitrogen) was used at a 1:10 dilution for 7 minutes, according to the manufacturers instructions. DNA was visualized using the mounting medium Prolong Gold with DAPI (Invitrogen). Transfection of siRNA and confirmation of knockdowns via western blots Pools of 4 targeted siRNAs were used simultaneously to independently knockdown β-Spectrin II, protein 4.1, α-adducin [20]. A www.selleckchem.com/products/Temsirolimus.html control pool of 4 non-targeting siRNAs (Dharmacon) was used to control for off target Selleck PFT�� effects. All transfections were performed using the InterferIN transfection reagent (PolyPlus Transfection), over a period of 48 hours, according to the manufactures instructions. The media was changed to standard DMEM with 10% FBS prior to the infections. Western blots were performed to confirm successful knockdown as outlined previously [20]. For assays that used siRNA-treated cells, the coverslips were examined microscopically, initially for cells that had complete knockdown of the protein of interest, then the number of bacteria in the cells were assessed by first confirming the bacteria were inside of the cells by scanning the samples from top to bottom and acquiringZ-stacks. Statistics Statistical analysis involved a 1-way ANOVA analysis, with Dunnett’s post-hoc test, to compare each

data set to the control group. When we compared data sets directly, we used a non-parametric student t-test. Acknowledgements Funding was provided by CIHR and NSERC. AEL is a CIHR CGS and a MSFHR click here awardee and JAG is a CIHR New Investigator. Electronic supplementary material Additional file 1: Figure S1 Modified Figure 1 with brightened actin. A modified version of Figure 1 with the actin levels brightened to show the actin in other regions of the host cell. This figure exemplifies how concentrated actin is at the site of S. flexneri infection. Scale bar is 5 μm (JPEG 532 KB) Additional file 2: Figure S2 RNAi images of S. flexneri infections showing non-transfected cells next to cells with near complete knockdown of spectrin, p4.1, or adducin. Spectrin, adducin, or p4.1 were knocked-down in HeLa cells prior to infection with S. flexneri for 1.