Peptides were bound to fluorescent latex beads, incubated with MAC-T bovine mammary epithelial cells, and internalization into MAC-T cells was evaluated using confocal laser and transmission electron microscopy.
Standards used in the gel filtration analyses (Fig. 1A) are "T": bovine thyroid thyroglobulin, diffusion coefficient D = 2.49×10−7 cm2s−1; "F": horse spleen ferritin, D = 3.61×10−7 Ca2s−1; "Ca": bovine liver catalase, D = 4.1×10−7 cm2s−1; "B": bovine serum albumin, D = 6.8×10−7 cm2s−1 (GE Healthcare).
Here, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157 H7 strains with varying Shiga toxin profiles from two common lineages.
Phenylmethylsulphonylfluoride (PMSF), polyethylinimine (PEI), kanamycin sulphate, thioflavin T (ThT), bovine κ-casein and bovine pancreas insulin were obtained from Sigma.
Van het Klooster et al., for instance, showed the formation of 2β, 6β, 11α, 12β, and 16α-OH-T when bovine hepatocyte cultures were exposed to T [ 24].
The whole-mounts were washed twice in PBS with 0.5% Triton-X100 (PBS-T) and incubated in blocking solution that consisted of PBS-T, 2% bovine serum albumin (BSA), and 10% non-immune goat serum for 1 h at room temperature.
In some experiments, nuclear extracts from MAC-T cells (bovine mammary epithelial cells; NEXIA Biotechnologies Inc).
As part of the validation of the assay for analysis of bovine T cells, we had shown that T-cell clones derived from T. parva-specific CD8+ T-cell lines were broadly representative of the uncloned parent cell line 29.
After 4 washes with TBS-T and blocking with TBS-T containing 2% bovine serum albumin (BSA), the viruses were probed with primary antibody, and then AP-conjugated secondary antibodies in TBS-T containing 0.5% BSA at room temperature for 2 hours, with extensive washes and blocking in between.
After permeabilization with PBS-0.1% Triton (PBS-T) and blocking with PBS-T containing 0.2% bovine serum albumin and 2% normal goat serum, larval fillets were incubated at 4°C overnight in solutions of primary antibody.
