Anti nfatc1 7a6

Recombinant human M‐CSF and human soluble RANKL were purchased from PeproTech (Rocky Hill, NJ, USA). Anti‐NFATc1 (7A6) and anti‐c‐Fos (H125) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and monoclonal antibodies against β‐actin (AC‐74) and secondary antibodies were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against ERK, JNK, p38, IκB, and Akt were from Cell Signaling Technology (Cambridge, MA, USA) as were phospho‐specific antibodies for ERK (Thr202/Tyr204), JNK (Thr182/Tyr185), p38 (Thr180/Tyr182), IκB (Ser32) and Akt (Ser473). All other reagents were obtained from Sigma‐Aldrich.

The cells were fixed in 4% formaldehyde and perm with 10% TritonX-100 for 15 min, washed three times with 0.01 M PBS, and incubated with 5% bovine serum albumin (Sigma-Aldrich) for 1 h for blocking. The sections were then incubated overnight at 4°C with anti-β-catenin (15B8, eBioscience) or anti-NFATc1 (7A6, Santa Cruz Biotechnology) primary antibodies. Next, the cells were washed three times with 0.01 M PBS, incubated with secondary antibody Alexa Fluor 488-conjugated Goat anti-Mouse IgG (#A11001, Invitrogen) for 4 h and washed three times with 0.01 M PBS. For nuclear counterstaining, the cells were incubated with 4′,6-diamidino-2-phenylindone (Cell Signaling Technology, #4083) for 10 min, washed in 1× PBS, and the slides were set with FluorSave™ reagent (EMD Millipore). Expression and localization of beta-catenin were observed with an Olympus FV1200 laser scanning confocal microscope (Olympus Corporation) with fixed exposure time for all samples. Colocalization was quantified as a ratio of overlapping pixels to the total number of pixels by Threshold Mander’s coefficient and expressed as nuclear percentage (% nuclear). The preparations were visualized using confocal microscopy (Olympus FV1200), and the analyses of images were carried out using FIJI/ImageJ.

Whole cell extracts of transfected HEK293 cells were prepared by lysing cells in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Cell Signaling Technology). The cells extracts were used for immunoprecipitation (IP), and immunoblotting (WB) was performed following the standard protocol^{3 (link),7 ,39 (link),40 (link)} using antibodies purchased from the following vendors: anti-MMP-13 (1:6000 dilution, Cat#39012, Abcam); anti-m-PD-1H and anti-h-PD-1H (1:2000 dilution, Cat# AF7005 and AF7126, R&D Systems); anti-h/m-PD-1H (for IP and WB) (1:50 dilution for IP; 1:2000 dilution for WB, Cat#54979, Cell Signaling Technology); anti-Flag tag (1:2000 dilution, Cat# 3165, Sigma-Aldrich); anti-c-myc tag (1:2000 dilution, Cat#sc-40, Santa Cruz Biotechnology); anti-DC-STAMP (clone 1A2) (1:2000 dilution, Cat#MABF39, Millipore); anti-NFATc1 (7A6) (1:2000 dilution, Cat#sc-7294, Santa Cruz biotechnology); anti-P-ERK1/2(D13.14.4E), and anti-ERK1/2, anti-P-c-Src (Y416), and anti-c-Src: (1:2000 dilution, Cat#4370; 9102; 2101; 2123; Cell Signaling Technology); and β-actin (1:6000 dilution, Cat#A5441, Sigma-Aldrich). Rac1 activation was determined using Active Rac1 Pull-Down and Detection Kit (Cat#16118, Thermo Fisher Scientific).