Na934

Protein cell lysates were harvested from primary osteoclasts in modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were cleared by centrifugation. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore). Blots were blocked in TBS/0.1% Tween-20 (TBST) plus 5% nonfat dry milk then incubated at 4°C overnight with primary antibodies diluted in TBST plus 5% bovine serum albumin. Primary antibodies used are included in Table 2. The next day, blots were washed with TBST, and then they were incubated for 1 hour at room temperature with horseradish-peroxidase conjugated secondary antibodies diluted in TBST plus 5% nonfat dry milk. Secondary antibodies used were from G.E. Health Systems: Amersham ECL anti-mouse (NA-931) and anti-rabbit (NA-934) at 1:6000, or Santa Cruz: anti-goat (SC-2020) at 1:6,000 dilution. Blots were then washed in TBST again before antibody binding was detected using a western blotting detection kit (Western Bright Quantum, Advansta) and the ChemiDoc Imaging System (Bio-Rad). Histone 3 (H3) or actin was used as a loading control for all blots.

Immunoblotting and co-immunoprecipitation experiments were performed as described (Hergovich et al., 2005 (link)). The characterization of Tao-1 and TAO1 binding to GCKIII kinases was carried out in low-stringency buffer as defined previously (Cook et al., 2014 (link)). Anti-HA antibodies were from Cell Signaling (C29F4) and Roche (3F10). Anti-myc antibodies were from Santa Cruz Technology (9E10) and Cell Signaling Technology (71D10). Anti-MST3 (611057) was from BD Biosciences, anti-MST4 (3822) was from Cell Signaling Technology, anti-STK25 (sc-6865) and anti-actin (sc-1616) were from Santa Cruz Biotechnology. anti-Mal (Maltose binding protein; E8032) is from New England BioLabs. The antibody detecting activation loop phosphorylation of GckIII (Thr167), MST3 (Thr190), MST4 (Thr178) and STK25 (Thr174) was from Abcam and termed anti-T167-P or anti-GCKIII-P, respectively. The antibody detecting hydrophobic motif phosphorylation of Trc (Thr449) has been described (Tamaskovic et al., 2003 (link)). Secondary antibodies were purchased from GE Healthcare (NA931, NA934, and NA935) and Santa Cruz Biotechnology (sc-2020).