Horseradish peroxidase conjugated secondary antibody against rabbit igg

This procedure has been described previously.24 ,25 Sixty micrograms of proteins from each sample was subjected to SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After 1-hour incubation in blocking buffer (Tris-buffered saline with 0.1% Tween and 5% nonfat dry milk), the membranes were incubated with a rabbit ZHX3 antibody (Abcam, Cambridge, MA, USA; dilution, 1:500) and a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Abcam; dilution, 1:1,000). Signals were captured with the enhanced chemiluminescence system following the manufacturer’s instructions (Amersham Pharmacia, Piscataway, NJ, USA). The membranes were reprobed with an anti-actin monoclonal antibody (Abcam; dilution, 1:1,000) to assure the equal loading of each sample. The intensity of ZHX3 was quantified using a Bio-Rad Quantity One quantitation software, with the ratio between the tumor and the paired noncancerous tissues of less than twofolds, suggesting downregulated ZHX3 expression.

The proteins were isolated from the adipocytes
using the Nuclear and Cytoplasmic Protein Extraction kit (Thermo Fisher
Scientific, MA, USA). A 10% SDS-polyacrylamide gel was used to separate
the proteins followed by transferring them to a polyvinylidene difluoride
membrane (Millipore, MA, USA). The membrane was incubated with 5%
nonfat dry milk in Tris-buffered saline/0.1% Tween-20, pH 7.4 for
1.5 h at room temperature and incubated overnight with primary antibodies
to PPAR-γ (Abcam, Cambridge, USA, 1:1000), C/EBP-α (Abcam,
Cambridge, USA, 1:1000), ATGL (Abcam, Cambridge, USA, 1:1000), AQP-7
(Abcam, Cambridge, USA, 1:1000), or GADPH (Abcam, Cambridge, USA,
1:1000). This was followed by incubation with a horseradish peroxidase-conjugated
secondary antibody against rabbit IgG (Abcam, Cambridge, USA, 1:5000).
Blots were incubated with the ECL reagents (Thermo Fisher Scientific,
MA, USA) and exposed to a Tanon 5200 Multi for the detection of the
protein expression. The software ImageJ was used to quantify the Western
blot results. First, we scanned the Western blot bands and subtracted
the backgrounds. We then selected the target bands and calculated
the integrated intensity of each band. Results were then exported
for statistical analysis.