Irdye 680rd goat anti mouse secondary antibody

N-terminally His6-tagged human calcineurin A (α isoform, truncated at residue 400), was expressed in tandem with the calcineurin B subunit in E.coli BL21 (DE3) cells (Invitrogen) and affinity purified using Ni-NTA-agarose with methods identical to the purification of His-tagged yeast calcineurin^{34 (link)}. A 16-amino-acid peptide corresponding to HoxB13 residues 255–270, or a mutant version with residues 260, 262, 264 and 265 changed to alanine, or a positive control (Saccharomyces cerevisiae Dig2 residues 95–116 containing a PxIxIT site) were fused to GST in vector pGX4T-3. Cell lysates from bacteria expressing the were obtained as in a previous study^{34 (link)}. Bacterial cell lysate (50–100 μg) was used to test co-purification with 2μg of purified His6-calcineurin as described^{34 (link)}. Co-purifying proteins were observed by western blotting with mouse GST (Bio Legend) and anti-His monoclonal primary antibodies and an IRDye680RD goat anti-mouse secondary antibody (LiCor) and imaged with the Li-Cor Odyssey imaging system.

Samples were homogenized in ice-cold water (20uL/mg) with the addition of protease inhibitors. Homogenates were centrifuged at 16,000 × g at 4°C for 10 minutes to remove insoluble material. The supernatant was collected and protein concentrations were determined by Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). For electrophoresis, samples were diluted 1:1 with sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 62.5 mM Tris-HCL, PH 6.8). Samples (15 µg protein) were denatured at 70°C for 10 min, separated by 15% SDS polyacrylamide gel electrophoresis, and transferred to PVDF membrane (Bio-Rad; Hercules, CA). Membranes were blocked in 50% blocking buffer (Li-Cor; Lincoln, NE)/50% PBS for 1 hr at room temperature, incubated in primary antibodies against MBP (1:2000; Millipore, Billerica, MA) or PLP (1:1000; Millipore) in blocking buffer/0.1% Tween-20 overnight at 4°C, washed (PBS, 0.05% Tween-20), incubated in IRDye^® 680RD goat anti-mouse secondary antibody (Li-Cor) for one hour, and washed again. Membranes were then scanned and bands were analyzed using an Odyssey^® infrared imager (Li-Cor). Membranes were also probed with an antibody against β-actin (1:5,000; Abcam, Cambridge, MA) as a loading control. Data are expressed as a ratio of the protein of interest and the corresponding density of β-actin.