Ab25916

Immunofluorescence in PDAC cells was performed as described previously [6 (link)], but using 13% or 4.5% of paraformaldehyde and utilizing the following primary and secondary antibodies: Myctag (Cell Signaling, #2272S, 1:200; Cell Signaling Technology, Danvers, MA, USA), HA (Cell Signaling, #2367, 1:100; Cell Signaling Technology, Danvers, MA, USA), pSer21EZH2 (Bethyl IHC-00388, 1:100; Bethyl, Montgomery, AL, USA), ALEXA Fluor 568 donkey anti-mouse (Invitrogen #A10037, 1:500; Carlsbad, CA, USA), and ALEXA Fluor 488 donkey anti-rabbit (Invitrogen #A32790, 1:500; Carlsbad, CA, USA). To assess the HA-NFATc1/pSer21EZH2 colocalization coefficient, 1228 DAPI-stained nuclei were counted in eleven IF pictures to determine the number of nuclei positive for both HA-NFATc1 and pSer21EZH2. H&E staining and IHC were performed as previously described [6 (link)]. Primary antibodies were: EZH2 (mouse: Cell Signaling #5246, 1:100; human: Leica NCL-L-EZH2, 1:300; Leica Biosystems Wetzlar, Germany), NFATc1 (abcam ab25916, 1:200; Cambridge, UK), and pSer21EZH2 (Bethyl IHC-00388, 1:50; Bethyl, Montgomery, AL, USA).

After conducting the micro-CT analysis, the femurs were preserved at 4°C for histological and immunohistochemical analyses. The right femurs of the 3 groups of mice were first decalcified in 15% ethylenediaminetetraacetic acid (EDTA, Sigma), embedded in paraffin blocks, and sectioned at a thickness of 5 μm using a microtome. Then, the sections were subjected to hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAcP) and immunohistochemical (IHC) staining. The primary antibodies against MMP9 (ab38898, Abcam), NFATc1 (ab25916, Abcam), Heme Oxygenase 1 (HO-1; ab13243, Abcam) and NOX1 (ab55831, Abcam) were used for immunohistochemistry. The stained sections were photographed using a Optical Microscope Olympus CX43 (Tokyo, Japan). The histomorphometric analyses of the bones were performed using the BIOQUANT OSTEO software (Bioquant Image Analysis Corporation, Nashville, TN, USA).

Proteins were isolated from various cultured PTECs and PCs, and protein levels were detected. Briefly, protein extracts were boiled in RIPA buffer (Beyotime, Shanghai, China) and separated by SDS-PAGE electrophoresis. Western blotting was performed with the antibodies against synaptopodin (syn) (1:1000, ABN481, Millipore) to detect its protein level in PCs. Anti-Bim antibodies (1:1000, ab32158, Abcam) were used to examine the HG-induced Bim expression and the inhibition effect on PTECs after transfection with the Lentiviral vector. Antibodies against NFAT2 (1:1000, ab25916, Abcam) were used to detect NFAT2 levels after transfection with siNFATc1 or p-NFATc1 into PTECs or PTEC-Bim-shRNA. Anti-MICAL2 antibodies (1:1000, 13965-1-AP, Proteintech) were used to detect the MICAL2 level in PCs after coculturing with un-transfected PTECs and PTEC-Bim-shRNA, and after transfection of NONHSAT179542.1 siRNA and siNC into PCs. β-actin (1:5000, 60008-1-Ig, Proteintech) was used as a reference protein.

After treatment with Uro-A, RIPA buffer (Beyotime) was used to lyse BMMs for harvesting total protein. We used SDS-polyacrylamide gels (Beyotime) to separate the proteins in the samples. Subsequently, proteins were transferred to PVDF membranes (Merck Millipore, MA, United States) and blocked with QuickBlock™ blocking buffer (Beyotime) for 1 h at room temperature. After incubation with primary antibodies overnight, PVDF membranes were rinsed three times with TBST (NCM Biotech, Suzhou). Then, the corresponding secondary antibodies (Beyotime) were added for another 1 h. We used an ECL reagent (Epizyme, Sigma–Aldrich) to detect antibody-antigen complexes. The following primary antibodies were used in our experiment: cathepsin K (CTSK; ab19027), nuclear factor of activated T cells 1 (NFATc1; ab25916), MMP9 (ab228402), ULK1 (ab203207), Beclin 1 (ab210498), LC3 (ab192890) and β-actin (ab6276), which were purchased from Abcam (Cambridge, United Kingdom). In addition, primary antibodies against ERK (#4695), phospho-ERK (#4370), JNK (#9252), phospho-JNK (#9255), P38 (#8690), and phospho-P38 (#4511) were provided by Cell Signaling Technology (Boston, United States).

Apical myocardia harvested from four to six hearts per treatment group were homogenized in a lysis buffer containing phosphatase and proteinase cocktail inhibitor, in a ratio of 100:1:1, respectively. Lysates of normalized concentrations treated with reducing agents were denatured at 100°C for 10 min and separated on SDS-PAGE gels. The transferred protein bands were blocked and immunoblotted overnight in the following primary antibodies: β₁AR (ab3442; Abcam), β₂AR (ab182136; Abcam), MEF2 (ab64644; Abcam), GRK5 (ab64943; Abcam) GATA4 (ab84593; Abcam), NFAT (ab25916; Abcam), AC5 (PAC-501AP; FabGennix), AC6 (PAC-601AP; FabGennix), AC7 (PAC-701AP; FabGennix), ANP (sc-515701; Santa Cruz Biotechnology), BNP (sc-271185; Santa Cruz Biotechnology), GRK2 (sc-13143; Santa Cruz Biotechnology), pNF-κB (3033T; Cell Signaling Technology), NF-κB (8242T; Cell Signaling Technology), Cleaved Caspase-3 (9661T; Cell Signaling Technology), Collagen Type I (14695-1-AP, Proteintech), Collagen Type III (13548-1-AP, Proteintech), and GAPDH (10494-1-AP; Proteintech). Immunoblots were performed in triplicates and normalized with their respective loading controls.