Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

Phosphorylated proteins were separated using SDS-PAGE (7.5–15% separating gel), transferred to membranes (1620177, BioRad) using a TransBlot Turbo Transfer System (1704150, BioRad), and blocked in 5% BSA (in TBST, A2153-1KG, Sigma-Aldrich). Blots were incubated overnight in rabbit anti-mouse primary antibody for phosphorylated protein of interest (Supplemental Table S5), then incubated in a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ab6721, Abcam). Phosphorylated proteins of interest were detected using Clarity Western ECL Blotting Substrate (1705061; BioRad). Images were captured using a ChemiDoc MP Imaging System (1708280; BioRad). Blots were incubated in stripping buffer (21059, ThermoFisher Scientific), washed twice in TBST, then blocked in 5% BSA (in TBST, pH 7.4, A2153; Sigma-Aldrich). Blots were probed with the rabbit anti-mouse primary antibody binding to the total protein (phosphorylated and unphosphorylated; Supplemental Table S5) protein of interest. Total protein was detected by incubating blots in Clarity Western ECL Blotting Substrate (1705061; BioRad). Images were captured using a ChemiDoc MP Imaging System (1708280; BioRad).

Western blot analysis was performed as described previously.^{19 (link)}
Briefly, cells used for western blot analysis were trypsinized and collected by centrifugation, and lysed in RIPA lysis buffer. Soluble proteins were collected and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and blocked with 5% skimmed milk for 1 hour. The membranes were then incubated with rabbit polyclonal antibody anti-SPOCD1 (Abcam, Cambridge, UK; #ab122188; 1:1000 dilution) or rabbit polyclonal antibody against GAPDH (Cell Signaling Technology, Cambridge, MA, USA; #2118; 1:4000 dilution) at 4°C for 2 hours. The immunoblots were washed twice with TBS containing 0.1% Tween 20 (TBST) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Abcam; #ab6721; 1:2000 dilution) at room temperature for 1 hour. After washing twice with TBST, the chemiluminescent signals on the immunoblots were visualized using a Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA; #170-5061) and imaged using a Tanon 5200 fully automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).

Immunohistochemical analysis was performed to detect CTCFL protein expression in the tissue specimens. Analysis revealed that CTCFL was the most upregulated gene in the four datasets and was subsequently selected for immunohistochemical analysis. Briefly, paraffin-embedded tissue blocks were cut into 4-µm thick sections and then placed in a constant temperature box at 65°C for 30 min to deparaffinize. The sections were submerged in the ethylenediaminetetraacetic acid buffer and microwaved for 8 min for antigenic retrieval. 3% hydrogen peroxide in methanol was used to quench the endogenous peroxidase activity. Then 1% goat serum albumin (Abcam) was incubated at room temperature for 5 min to block nonspecific binding. The sections were subsequently stained with an anti-BORIS (CTCFL) primary antibody (1:200; cat. no. ab187163; Abcam) and incubated overnight at 4°C, and horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:1,000; cat. no. ab6721; Abcam) were then incubated at room temperature for 1 h. Finally, images were captured using a high-capacity digital slide scanner (Pannoramic SCAN, 3DHISTECH) at ×200 magnification. The sections were evaluated independently by two experienced pathologists. A total of 12 patients with paired serous ovarian cancer patients, were selected from patients enrolled in our study to conduct this experiment.