Sc 74543

The immunofluorescence procedures were performed according to our previous publication [32 (link)]. Briefly, the C6, C7, and C8 spinal segments of each rat were removed carefully, immersed in fixative (4% paraformaldehyde solution) for about 3–4 h, and then suspended in 30% (v/v) sucrose solution in phosphate-buffered saline (PBS) until they sank to the bottom of the tube. Frozen transverse sections (35μm) were washed three times with 0.01M PBS for 10 min each time and incubated in 3% BSA 0.01M PBS at room temperature for 1 h. The slices were then incubated overnight at 4 °C with the following primary antibodies: Santa Cruz #3270, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) c-Jun (1:200, Santa Cruz sc-74543), nNOS (1:200, Santa Cruz #4231), Glial fibrillary acidic protein (GFAP) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA #sc-51908). After washing in PBS, sections were then incubated with respective secondary antibodies at room temperature for 1 h. The slices were then washed thrice, mounted on glass slides, and their images were captured using a Nikon Olympus BX-63 microscope. The staining specificity was tested by the omission of primary antibodies.

To prevent interference from Dmp1-GFP fluorescence in IDG-SW3 cells after mineralization induction, we used unmineralized IDG-SW3 cells for IF staining. Osteocytes were fixed with cold 4% paraformaldehyde for 15 min, treated with 0.2% Triton for 5 min, blocked with 10% goat serum at room temperature for 60 min, and finally incubated with primary antibodies against c-JUN (Santa Cruz Biotechnology, sc-74543; mouse mAb, dilution 1:50) and NRF2 (CST, 12721; rabbit mAb, dilution 1:200) at 4 °C for 16 h. The next day, the samples were washed 3 times with TBST and incubated with anti-mouse IgG (CST; Alexa Fluor 594, dilution 1:1 000) and anti-rabbit IgG (CST; Alexa Fluor 488, dilution 1:1 000) secondary antibodies at room temperature for 60 min. Then, nuclei were stained with DAPI solution for 10 min. Confocal laser scanning microscopy was used for image acquisition.

HEK293T cells were transfected with pcDNA3 encoding FLAG-tagged JunB, c-Jun, or JunD to express substantial amounts of these proteins. FLAG–JunB, FLAG–c-Jun or FLAG–JunD in the cell lysates were used as standard proteins in immunoblot analysis for estimation of relative amounts of endogenous JunB, c-Jun, and JunD in Th17 cells. The same amounts of FLAG–JunB, FLAG–c-Jun or FLAG–JunD, estimated by immunoblot using the anti-FLAG antibody (M2, Sigma), were serially diluted and subjected to immunoblot analysis; in the analysis, antibodies specific for JunB (sc-46, Santa Cruz), c-Jun (sc-74543, Santa Cruz), or JunD (sc-74, Santa Cruz) were used for estimation of the corresponding endogenous Jun protein in the Th17 cell lysate (see Fig. 4a). These primary antibodies were probed with the fluorescently labeled secondary antibody, anti-mouse IRDye800CW-conjugated (LI-COR Biosciences). Detection and quantification were performed using the Odyssey Infrared Imaging System (LI-COR Biosciences)^{58 (link),59 (link)}. Standard curves were generated by plotting the band intensities against the amounts of FLAG-tagged Jun-family proteins, and used for calculation of the relative expression level of endogenous JunB, c-Jun, and JunD in Th17 cells (see supplemental Fig. 5).