Ab207434

Cells were cultured in complete DMEM till 70–80% confluence and serum starved for 5 h before stimulation. Medium was replaced with complete medium with or without recombinant mouse IL-22 (50 ng/ml, eBioscience) for 10 min stimulation. Cells were then washed with 1x PBS and immediately scraped from the plate into 1x RIPA lysis buffer containing protease/ phosphatase inhibitors (Thermo Fisher). The lysates were then sonicated and centrifuged at 14,000 g for 15 min and the supernatants were collected and quantified with a Bio-rad protein assay kit for Western blot analysis. Protein samples were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride membrane. Membranes were blocked with 5% skim dried milk in Tris buffered saline buffer (TBST: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and incubated overnight with the following primary antibodies: Anti-ATF3 (Abcam ab207434), pSTAT3 (CST#9145), STAT3 (CST#9139), PTP-SHP-2 (Santa Cruz #sc-7384), PTP-MEG2 (#sc-2271052), PAC-1 (#sc-32776), β-actin (#sc-47778), Histone H3 (CST#4499), GAPDH (CST#5174), α-tubulin (#abcam2791, Clone DM1A). After washing with TBST, blots were incubated with HRP conjugated secondary antibodies. Detection was achieved using a chemiluminescence substrate (Omics Bio) and images were acquired using ImageQuant™ LAS 4,000 camera.

Proteins were extracted using TRIzol reagent (Invitrogen) and resolved in 1% SDS. After SDS‐PAGE, the samples were transferred to nitrocellulose membranes and incubated for 30 min at room temperature with 5% skimmed milk. The membranes were incubated overnight at 4°C with specific primary antibodies: anti‐Lamin B1 (1:1000, 12987‐1‐AP, Proteintech), anti‐β‐actin (1:2000, sc‐47778, Santa Cruz Biotechnology), anti‐p16 (1:500, ab108349, Abcam), anti‐ATF3 (1:500, ab207434, Abcam), and anti‐Tublin (1:1000, T6199, Sigma). After washing three times with PBST (1× PBS and 0.1% Tween‐20), the membranes were incubated at room temperature for 2 h with IRDye 800CW Goat/Donkey anti‐Mouse/Rabbit secondary antibodies (1:10,000, 926‐32210; LI‐COR Biosciences). We acquired blot information using the Odyssey Infrared Imaging System (LI‐COR).

Five hundred thousand B16-F10 cells were seeded in the circle microscope cover glasses and cultured overnight. CBD (Ca²⁺ (high)) at a CBD concentration of 0, 2.5, and 4 µg/mL were co-incubated with the cells for 2 h. After the removal of the medium, the cover glasses were fixed with 4% paraformaldehyde. Then, the cells were then permeabilized with Triton X-100 (0.1%, v%) for 15 min, followed by the addition of a blocking solution consisting of bovine serum albumin (BSA, 1%) and incubated at room temperature for 30 min. Next, all samples were incubated with the primary antibodies at room temperature for 12 h, followed by staining with the corresponding secondary antibodies in dark for 1 h. The primary antibodies included ATF3(ab207434, Abcam) and NFATc1(sc-7294, SANTA CRUZ). The following secondary antibodies included IgG (H + L) Fluor 555-conjugated (A0460, Beyotime) and IgG (H + L) Fluor 647-conjugated (A0468, Beyotime). Nuclei were labeled with DAPI (BL105A, Biosharp) at room temperature for 5 min. After washing with PBS thoroughly, immunofluorescence images were then acquired with a multiphoton confocal microscopy (STELLARIS 8 DIVE, Leica).

Cells were lysed in ice-cold RIPA buffer containing protease inhibitors. Protein samples (40 µg) were loaded for electrophoresis on 8–12% denaturing SDS-PAGE gels. The blots were probed with the corresponding primary antibodies overnight at 4 °C, followed by incubation with the appropriate secondary antibodies at room temperature for 1 h. The following primary antibodies were used for detection: ATF3 (1:2000, #ab207434, Abcam. Inc), Twist1(1:2000, #25465-1-AP, Proteintech. Inc), Slug (1:2000, #12129-1-AP, Proteintech. Inc), Snai1(1:2000, #13099-1-AP, Proteintech. Inc), and GAPDH (1:2000, # YM3029, Immunoway. Inc).

70% confluence hESCs were treated with Ad-ATF3-His or Ad-LacZ for 2 days. Crosslink, cell lysis, genomic DNA fragments extraction were performed as described^{32 (link)}, and then immunoprecipitated using ATF3 antibody (ChIP Grade, ab207434, Abcam) and nonspecific IgG as technical control. The specific primers that were used to amplify the hsa-miR-135b promoter DNA fragments containing a putative ATF3 binding sequence were 5′-CAGCTGAAGCCCTCTTTCTG-3′ and 5′-AGGAGGGTCTGGGTAAAGGA-3′.