Sheep α mouse hrp

A549 cells were transfected according to manufacturer’s instructions (Dharmafect 1; Thermo Scientific) with ON-TARGET plus smart pool siRNA targeting RelA, human TLR5 or a non-targeting control 24 h prior to treating with flagellin and pp infection. Silencing was assessed by PCR/western blotting. RNA was prepared using the Qiagen RNeasy kit and amplified for TLR5 using human TLR5 primer pair Hs01019558_m1 (Thermo Scientific) in a quantitative reverse-transcription PCR (qRT-PCR) in accordance with the manufacturer’s guidelines (CellsDirect kit; Thermo Scientific) and fluorescence monitored in a 7500 real-time PCR machine (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included as an endogenous control for amplification efficiency, and TLR5 amplification normalized to GAPDH using the ΔΔCt method. Proteins were separated using an 8% SDS-PAGE gel, transferred to PVDF membrane (Millipore) and probed using a primary RelA (p65) rabbit monoclonal antibody (Cell Signaling Technology) or α-actin mouse monoclonal antibody (Sigma Aldrich) and secondary donkey α-rabbit HRP antibody (GE Healthcare) or sheep α-mouse HRP (GE Healthcare). Membranes were incubated in EZ-ECL prior to image capture using a PXi Multi-application gel imaging system (Syngene).

The monoclonal mouse-α-GP1 H3C8 antibody was a kind gift from Carolyn Wilson of the Food and Drug Administration. Rat-α-HA-HRP 3F10, rabbit α-GAPDH and mouse-α-beta tubulin were from Sigma Aldrich. The monoclonal rabbit-α-NPC1 (ab134113) was from Abcam. The anti-beta tubulin mouse monoclonal antibody was from Sigma Aldrich. The sheep-α-mouse-HRP, the donkey-α-rabbit-HRP and the streptavidin magnetic sepharose beads were from GE Healthcare. The α-rabbit IR800, α-mouse IR680, and α-rat IR680 antibodies were from Licor. The mouse α-VSV-M antibody (23H2) was a gift from Michael Whitt.