Immobilon polyvinylidene difluoride membrane

Control and IL-1β (50 ng/mL)-treated SH-SY5Y cells were lysed in radioimmunoprecipitation assay buffer (Sigma) in the presence of a protease inhibitor cocktail (Roche, Branchburg, NJ). The soluble protein fraction was collected after centrifugation (14,000 rpm, 20 min) and quantified using the Dc protein kit (Bio-Rad). An equal amount of protein (40 μg) was loaded onto 4–12% bis-Tris gradient mini gels (Invitrogen) and transferred onto Immobilon polyvinylidene difluoride membrane (Fisher Scientific). The blots were then probed with anti-THTR-1 (1:1000), anti-Sp1 (1:500), and anti-β-actin (1:3000). The specificity of the THTR-1 polyclonal antibodies has been validated by us in previous studies (Ramamoorthy et al., 2020 (link)). The immune-reactive bands were detected with the corresponding secondary antibodies anti-rabbit IRDye-800 and anti-mouse IRDye-680 (both at 1: 30,000 dilution). The densities of the immune-reactive bands were quantified using LI-COR software (LI-COR Bioscience, Lincoln, NE).

EHEC infected and uninfected NCM460 cells and mouse colon tissues were lysed in radio immunoprecipitation assay buffer (RIPA buffer; Sigma) containing complete protease inhibitor cocktail (Roche, NJ). The soluble protein fraction was collected followed by centrifugation (12,000 rpm, 20 min). Concentrations of proteins were determined using Dc protein assay kit (Bio-Rad). To determine the level of TPP transporter and transcription factors protein expression, an equal amount of protein (40 μg) was loaded onto 4–12% Bis-Tris gradient minigels (Invitrogen) and transferred in to Immobilon polyvinylidene difluoride membrane (Fisher Scientific). Subsequently, the blots were probed with anti-TPPT (1:200), anti-CREB-1 (1:200), anti-ELF3 (1: 200), anti-ERK1/2 (1:1000), anti-Phospho ERK1/2 (1:1000), anti-Phospho NF-κB (1:1000), anti-NF-κB (1:1000), anti-β-actin (1:3000), and anti-Lamin B (1: 1000) antibodies. The membranes were probed with anti-mTPPT antibody and anti-mTPPT antibody pretreated with antigenic peptide to show the specificity of the mTPPT antibody. Anti-rabbit IRDye-800 and anti-mouse IRDye-680 (both at 1:30,000 dilutions) were used as the secondary antibodies in this study. The specific immunoreactive bands were detected using the Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE), and their densities were measured using the LI-COR software.