Hrp conjugated anti rabbit secondary antibody

Individual clones were grown until 100 % confluency and collected for dot blot analysis, dislodging the monolayer of cells by pipeting within the well. 90 µl of the 100 µl total volume was then removed, spun down, and the cell pellet lysed in 10 µl of 1× Passive Lysis Buffer (Promega’s Dual-Luciferase Reporter Assay System, Cat: E1910). The remainder of the cells were propagated. 1 µl of cell lysate was pipetted onto dry nitrocellulose membrane (Bio-Rad, Cat: 162-0115) to form a dot. Each sample was blotted twice on two separate membranes, creating two identical patterns of samples, blocked in 5 % milk in TBST for 1 h at room temperature, and blotted for HuR (Santa Cruz Biotechnology, clone 3A2, Cat: sc-5261, 1:1000–1:5000 dilution) and Pum2 (Bethyl Labs, Cat: A300-202A, 1:1000 dilution) in 5 % milk in TBST for 1 h, with 3 × 5 min TBST washes in between incubation steps. Anti-Mouse-HRP-conjugated secondary antibody was from Cell Signaling (anti-mouse, Cat: 7076). Anti-Rabbit-HRP-Conjugated Secondary Antibody was from Cell Signaling (anti-rabbit, Cat: 7074). Blotted membranes were imaged on a Biorad ChemiDoc MP imager and quantified by Image Lab: identical circles were centered on each dot, and the signal volume was computed by the software. Local background values for each dot were computed and subtracted by Image Lab. Western blotting was performed as described above.

Cell lysates for Western blotting were prepared as previously described [17 (link)] with minor modifications. Briefly, HCT116, HT-29, and Caco-2 cells in 60 mm culture dishes were treated with butyrate (0 to 4 mM) when cells reached ~30–40% confluency. Cells were harvested and lysed 24 h after butyrate treatment using cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Bradford assay was performed to determine the protein concentration. Protein samples were separated with SDS-page gel electrophoresis using gradient gels (4 to 20%) and transferred onto PVDF membranes. After blocking the membranes with 5% dry milk, membranes were incubated overnight at 4 °C with the following primary antibodies: phospho-p44-42 (ERK1/2) T-202/Y-204, ERK1/2, p21, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc., Danvers, MA, USA), and c-Myc (Abcam, Cambridge, MA, USA). After TBS wash, membranes were incubated for 1 h with anti-rabbit HRP-conjugated secondary antibody (1:5000 dilution) (Cell Signaling Technology, Inc., Danvers, MA, USA). Finally, these membranes were TBS-washed and then incubated with chemiluminescence (ECL) reagent; and protein images were visualized and quantified using a LI-COR Odyssey Fc imager system (Lincoln, NE, USA).