Anti myc clone 9b11

Transfected Tcons after incubation were stimulated and washed with PBS as described earlier, and lysed in Beadlyte Cell Signaling Universal Lysis Buffer (Upstate) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, cat. no. 78440). Proteins were denatured in SDS sample buffer, resolved by SDS-PAGE using 10% Mini-PROTEAN TGX Gel (Bio-Rad, cat. no. 4561035S), and transferred to Protran nitrocellulose membranes (Amersham GE Healthcare, cat. no. 10600002). Afterward, the membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% (w/v) Tween 20 (TBST) and incubated with primary (see below) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). Protein bands were developed with Immobilon Western Chemiluminescent HRP substrate (Millipore) in a Vilber Fusion Solo S chemiluminescence acquisition system (Vilber Lourmat). Bands were quantified using the ImageJ software version 1.5Oi. Anti-NFAT1 (clone 4G6-G5, Santa Cruz Biotechnology, cat. no. sc-7296), anti-myc (clone 9B11, Cell Signaling Technology, cat. no. 2276), anti-DEF6 (clone EPR7492, Abcam, cat. no. ab126792), and anti-GAPDH (clone 6C5, Santa Cruz Biotechnology, cat. no sc-32233) antibodies were used for Western blot to detect corresponding targets.

For the determination of endogenous LGR6 expression, C2C12 myoblasts were cultured in a differentiation medium. Cells were then sonicated in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM DTT, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium molybdate, and 10 mM sodium pyrophosphate) to prepare cell lysates. For the pull-down assay, the cell lysates were incubated with Ni-Sepharose 6 Fast Flow resin for 2 h at 4 °C, followed by washing with lysis buffer. Cell lysates (input) and resin-bound proteins were prepared. Proteins were subjected to SDS–PAGE, followed by Western blot analysis using the following primary antibodies: rabbit monoclonal anti-LGR6 (clone EPR6874; Abcam, Cambridge, UK), mouse monoclonal anti-β-actin (clone 2D4H5; Proteintech Group, Inc., Rosemont, IL, USA), anti-Myc (clone 9B11; Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal anti-ubiquitin (Cell Signaling Technology) antibodies. Horseradish-peroxidase-conjugated goat anti-mouse or anti-rabbit IgG was used as a secondary antibody. The blots were treated with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).