Ab22554

Protein preparation, SDS-PAGE, and western blotting were performed as previously described^{27 (link)}. Anti-CDKAL1, anti-β-catenin and anti-adiponectin (19F1) antibodies were purchased from Abcam (Cambridge, UK) (ab169531, ab32572 and ab22554). Anti-β-actin and anti-PPARγ antibodies were purchased from Santa Cruz Biotechnology (TX, USA) (sc-1616R and sc-7196). Anti-Lamin A/C antibody, anti-GSK3-β and anti-phopho-GSK-3β were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan) (#2032 s, #9315 and #9336). Anti-non-phospho (active) β-catenin antibody was purchased from Merck Millipore (Darmstadt, Germany) (#05-665). Peroxidase-linked anti-rabbit or mouse IgG secondary antibody was purchased from ThermoFisher Scientific (Yokohama, Japan) (#65-6120 and A10668).

Subcutaneous white adipose tissues (SAT) were isolated and immediately fixed with 4 % paraformaldehyde, then embedded into paraffin. Immunohistochemical analyses were performed for examining adiponectin expressions as described by Schindler et al. [38 (link)]. Tissue sections were first deparaffinized, and subjected to antigen retrieval at 95 °C for 15 min in antigen retrieval buffer (ab93680; Abcam, Cambridge, Massachusetts). Endogenous peroxidase activity was blocked with 0.3 % H₂O₂ in 80 % methanol for 30 min. The sections were then blocked with 1 % BSA and incubated with an anti-mouse adiponectin antibody (ab22554; Abcam, Cambridge, Massachusetts) for 18 h at 4 °C. Subsequently, the sections were incubated with a goat anti-mouse secondary antibody (K500711, DakoCytomation Ltd., CA, USA) for 30 min and visualized using a DakoCytomation EnVision® + Duallink system HRP (DAB+) kit (K4065, DakoCytomation Ltd., CA, USA). Slides were counterstained with hematoxylin, rinsed in ddH₂O for 10 min, and mounted with glycerin gelatin (GG1, Sigma-Aldrich Co., St. Louis, Missouri, USA). Representative 40X images were photographed and presented. Five areas of a section were used for the signal intensity quantification. The signal intensity of adiponectin expression in SAT was calculated using Image J, an image-analysis program (NIH, Bethesda, MD, USA) [39 ].

Cells were lysed in Proprep solution (Intron, Daejeon, Korea). Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, and incubated with appropriate antibodies. The following primary antibodies were AdipoR1, AdipoR2, APPL1 (rabbit monoclonal, Cell Signaling 3858, 1:1000), AMPKα (rabbit polyclonal, Cell Signaling 2532, 1:1000), phospho-AMPKα (rabbit polyclonal, Cell Signaling 2531, 1:1000), Akt (rabbit monoclonal, Cell Signaling 4691, 1:1000), phospho-Akt (rabbit polyclonal, Cell Signaling 9271, 1:1000), SREBP-1, adiponectin (mouse monoclonal, Abcam ab22554, 1:1000), and actin(goat polyclonal, Santa Cruz SC-1615, 1:1000) as a loading control. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Intron). Protein intensity was quantified using IMT i-Solution software (IMT Inc., Daejeon, Korea), and the values obtained were normalized relative to the actin signal.

Cells were cultured on disks at specific time points. Media was aspirated, and disks were washed in PBS. Cells were fixed with 4% PFA and permeabilized, incubated in blocking buffer, and then antibodies against adiponectin (Abcam, ab22554), vinculin (Abcam, ab18058), or YAP (SCBT, sc101199) overnight. Disks were washed, incubated in the appropriate second antibody for one hour, and mounted on glass slides for microcopy using a fluorescent microscope. Immunofluorescence images for adipogenesis and osteogenesis were quantified using MetaMorph. Vinculin and YAP were imaged with a fluorescent microscope and confocal microscope (Leica). YAP was manually quantified as predominantly nuclear versus cytoplasmic^{33 (link)}.

Anti-adiponectin antibody (Abcam, USA; ab22554) was used for immunostaining of BM-LMCs with a 1/400 dilution. The wells were firstly washed with PBS and then incubated with 4% of cold paraformaldehyde for 10 followed by 5 min at RT. Triton X-100 0.25% was added for 20 min at RT. After washing with PBS (Gibco), H₂O₂ 0.3% was added for 20 min in a dark place at RT followed by two times washing with PBS. The cells were incubated with 5% goat serum (Sigma) for 45 min at RT. After removing goat serum, the first antibody diluted in bovine serum albumin (BSA) (Sigma)/PBS 0.1% was added and the plate was kept at 4 °C overnight. Then, the wells were washed with PBS–Tween 0.1%, 3 × 5 min. After incubation with BSA/PBS 1% for 30 min at RT, the second antibody (Abcam, ab205719, 1/1000) was added to wells for 3 h. Subsequently, the wells were washed with PBS–Tween 0.1%, 3 × 5 min. 3,3'-Diaminobenzidine (DAB) (Sigma, Germany) solution was used as chromogen on plates for 10 min at RT. The wells were washed with PBS, 2 × 5 min. Finally, PBS was added to the wells.

Immunohistochemistry was performed as previously described [36 ]. Anti-adiponectin antibody (Abcam, ab22554, IHC: 1/400, mouse) and anti-GFP antibody (Santa Cruz, sc-390394, IHC: 1/200) were used in this study. For quantification of immunohistochemistry (IHC) results, ten random areas were captured for each day and the DAB-stained regions were quantified using the IHC Toolbox Plugin of ImageJ software.